bioRxiv Channel: University of Illinois Chicago

Binding of Calcium and Magnesium to Cardiac Troponin C

Rayani, K. — 2020-06-15

Cardiac troponin C (cTnC) is the Ca2+-sensing component of the thin filament. It contains structural sites (III/IV) which bind both Ca2+ and Mg2+, and a regulatory site (II) that has been thought to bind only Ca2+. The latter binding initiates a series of conformational changes that culminate in force production. We have quantified the interaction between site II and Ca2+/Mg2+ through Isothermal Titration Calorimetry and Thermodynamic Integration simulations. Direct and competitive binding titrations using wild type and a double mutant that significantly reduces binding to site II demonstrated that physiologically relevant concentrations of both Ca2+/Mg2+ interact with the same locus. Cytosolic free Mg2+ (~1 mM) could occupy a significant population of available site II, as this concentration of Mg2+ decreased the affinity for Ca2+ 1.4-fold. Interaction of Mg2+ with site II of cTnC likely has important functional consequences for the heart at baseline and in diseased states which decrease or increase availability of Mg2+ such as secondary hyperparathyroidism or ischemia, respectively.

Unique functions for Notch4 in murine embryonic lymphangiogenesis

Muley, A. — 2021-02-20

In mice, embryonic dermal lymphatic development is well-understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4 and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts, and differentially and temporally regulated the expression of Notch1 and Hes/Hey genes. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5, and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increased in LEC proliferation, described for the loss of LEC Notch1. Moreover, loss of Notch4 did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.

Cannabidiol Inhibits SARS-CoV-2 Replication and Promotes the Host Innate Immune Response

Nguyen, L. C. — 2021-03-10

The rapid spread of COVID-19 underscores the need for new treatments. Here we report that cannabidiol (CBD), a compound produced by the cannabis plant, inhibits SARS-CoV-2 infection. CBD and its metabolite, 7-OH-CBD, but not congeneric cannabinoids, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after cellular infection, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD induces interferon expression and up-regulates its antiviral signaling pathway. A cohort of human patients previously taking CBD had significantly lower SARS-CoV-2 infection incidence of up to an order of magnitude relative to matched pairs or the general population. This study highlights CBD, and its active metabolite, 7-OH-CBD, as potential preventative agents and therapeutic treatments for SARS-CoV-2 at early stages of infection.

Structural Covariance Networks in Post-Traumatic Stress Disorder: A Multisite ENIGMA-PGC Study

Rakesh, G. — 2021-03-16

IntroductionCortical thickness (CT) and surface area (SA) are established biomarkers of brain pathology in posttraumatic stress disorder (PTSD). Structural covariance networks (SCN) constructed from CT and SA may represent developmental associations, or unique interactions between brain regions, possibly influenced by a common causal antecedent. The ENIGMA-PGC PTSD Working Group aggregated PTSD and control subjects data from 29 cohorts in five countries (n=3439). MethodsUsing Destrieux Atlas, we built SCNs and compared centrality measures between PTSD subjects and controls. Centrality is a graph theory measure derived using SCN. ResultsNotable nodes with higher CT-based centrality in PTSD compared to controls were left fusiform gyrus, left superior temporal gyrus, and right inferior temporal gyrus. We found sex-based centrality differences in bilateral frontal lobe regions, left anterior cingulate, left superior occipital cortex and right ventromedial prefrontal cortex (vmPFC). Comorbid PTSD and MDD showed higher CT-based centrality in the right anterior cingulate gyrus, right parahippocampal gyrus and lower SA-based centrality in left insular gyrus. ConclusionUnlike previous studies with smaller sample sizes ([≤]318), our study found differences in centrality measures using a sample size of 3439 subjects. This is the first cross-sectional study to examine SCN interactions with age, sex, and comorbid MDD. Although limited to group level inferences, centrality measures offer insights into a nodes relationship to the entire functional connectome unlike approaches like seed-based connectivity or independent component analysis. Nodes having higher centrality have greater structural or functional connections, lending them invaluable for translational treatments like neuromodulation.

In BRCA1 and BRCA2 breast cancers, chromosome breaks occur near herpes tumor virus sequences

Friedenson, B. A. — 2021-04-20

Inherited mutations in BRCA1 and BRCA2 genes increase risks for breast, ovarian, and other cancers. Both genes encode proteins for accurately repairing chromosome breaks. If mutations inactivate this function, broken chromosome fragments get lost or reattach indiscriminately. These mistakes are characteristic of hereditary breast cancer. We tested the hypothesis that mistakes in reattaching broken chromosomes preferentially occur near viral sequences on human chromosomes. We tested millions of DNA bases around breast cancer breakpoints for similarities to all known viral DNA. DNA around breakpoints often closely matched the Epstein-Barr virus (EBV) tumor variants HKHD40 and HKNPC60. Almost all breakpoints were near EBV anchor sites, EBV tumor variant homologies, and EBV-associated regulatory marks. On chromosome 2, EBV binding sites accounted for 90% of breakpoints (p<0.0001). On chromosome 4, 51/52 inter-chromosomal breakpoints were close to EBV variant sequences. Five viral anchor sites at critical genes were near breast cancer breakpoints. Twenty-five breast cancer breakpoints were within 1.25% of breakpoints in model EBV cancers. EBV-like sequence patterns around breast cancer breakpoints resemble gene fusion breakpoints in model EBV cancers. All BRCA1 and BRCA2 breast cancers had mutated genes essential for immune responses. Because of this immune compromise, herpes viruses can attach and produce nucleases that break chromosomes. Alternatively, anchored viruses can retard break repairs, whatever the causes. The results imply proactive treatment and prevention of herpes viral infections may benefit BRCA mutation carriers.

A SARS CoV-2 nucleocapsid vaccine protects against distal viral dissemination

Class, J. — 2021-04-26

The SARS CoV-2 pandemic has killed millions of people. This viral infection can also result in substantial morbidity, including respiratory insufficiency and neurological manifestations, such as loss of smell and psychiatric diseases. Most SARS CoV-2 vaccines are based on the spike antigen, and although they have shown extraordinary efficacy at preventing severe lung disease and death, they do not always confer sterilizing immune protection. We performed studies in K18-hACE2 mice to evaluate whether the efficacy of SARS CoV-2 vaccines could be augmented by incorporating nucleocapsid as a vaccine antigen. We vaccinated mice with adenovirus-based vaccines encoding spike antigen alone, nucleocapsid antigen alone, or combined spike and nucleocapsid antigens. Mice were then challenged intranasally with SARS CoV-2, and acute viral loads were quantified at a proximal site of infection (lung) and a distal site of infection (brain). Interestingly, the spike-based vaccine conferred acute protection in the lung, but not in the brain. The spike-based vaccine conferred acute protection in the brain only if combined with the nucleocapsid-based vaccine. These findings suggest that nucleocapsid-specific immunity is important for the distal control of SARS CoV-2, warranting the inclusion of nucleocapsid in next-generation COVID-19 vaccines.

Sex-Specific Plasticity Explains Genetic Variation in Sexual Size Dimorphism in Drosophila

Vea, I. — 2021-06-17

The difference in body size between females and males, or sexual size dimorphism (SSD), is almost ubiquitous, and yet we have a remarkably poor understanding of the developmental-genetic mechanisms that generate it. Such an understanding is important if we are to distinguish between the many theoretical models of SSD evolution. One such model is the condition dependence hypothesis, which proposes that the body size of the larger sex is also more environmentally sensitive, a phenomenon called sex-specific plasticity (SSP). Because SSP generates differences in female and male body size, selection on plasticity may underlie the evolution of sexual size dimorphism. To test this hypothesis, however, we need to know the genetic architecture of both SSD and SSP, which is challenging because both are characteristics of populations not individuals. Here, we overcome this challenge by using isogenic lineages of Drosophila to measure both SSD and SSP for a genotype. We demonstrate extensive genetic variation for SSD among genotypes that is tightly correlated with variation in SSP, indicating that the same developmental-genetic mechanisms regulate both phenomena. These data support the condition dependence hypothesis and suggest that the observed SSD is a consequence of selection on the developmental-genetic mechanisms that regulate SSP.

A steroid hormone regulates growth in response to oxygen availability

Kapali, G. — 2021-06-25

In almost all animals, physiologically low oxygen (hypoxia) during development slows growth and reduces adult body size1-3. The developmental mechanisms that determine growth under hypoxic conditions are, however, poorly understood. One hypothesis is that the effect of hypoxia on growth and final body size is a non-adaptive consequence of the cell-autonomous effects of hypoxia on cellular metabolism. Alternatively, the effect may be an adaptive coordinated response mediated through systemic physiological mechanisms. Here we show that the growth and body size response to moderate hypoxia (10% O2) in Drosophila melanogaster is systemically regulated via the steroid hormone ecdysone, acting partially through the insulin-binding protein Imp-L2. Ecdysone is necessary to reduce growth in response to hypoxia: hypoxic growth suppression is ameliorated when ecdysone synthesis is inhibited. This hypoxia-suppression of growth is mediated by the insulin/IGF-signaling (IIS) pathway. Hypoxia reduces systemic IIS activity and the hypoxic growth-response is eliminated in larvae with suppressed IIS. Further, loss of Imp-L2, an ecdysone-response gene that suppresses systemic IIS, significantly reduces the negative effect of hypoxia on final body size. Collectively, these data indicate that growth suppression in hypoxic Drosophila larvae is accomplished by systemic endocrine mechanisms rather than direct suppression of tissue aerobic metabolism.

AFA: Computationally efficient Ancestral Frequency estimation in Admixed populations: the Hispanic Community Health Study/Study of Latinos

Granot Hershkovitz, E. — 2021-08-11

We developed a computationally efficient method, Ancestral Frequency estimation in Admixed populations (AFA), to estimate the frequencies of bi-allelic variants in admixed populations with an unlimited number of ancestries. AFA uses maximum likelihood estimation by modeling the conditional probability of having an allele given proportions of genetic ancestries. It can be applied using either global or local proportions of genetic ancestries. Simulations mimicking admixture demonstrated the high accuracy of the method. We implemented the method on data from the Hispanic Community Health Study/Study of Latinos (HCHS/SOL), an admixed population with three predominant continental ancestries: Amerindian, European, and African. Comparison of the European and African estimated frequencies to the respective gnomAD frequencies demonstrated high correlations, with Pearson R2=0.97-0.99. We provide a genome-wide dataset of the estimated three ancestral allele frequencies in HCHS/SOL for all available variants with allele frequency between 5%-95% in at least one of the three ancestral populations.

In the Line of Fire: Debris Throwing by Wild Octopuses

Godfrey-Smith, P. — 2021-08-19

Wild octopuses at an Australian site frequently propel shells, silt, and algae through the water by releasing these materials from their arms while creating a forceful jet from the siphon held under the arm web. These "throws" occur in several contexts, including interactions with conspecifics, and material thrown in conspecific contexts frequently hits other octopuses. Some throws appear to be targeted on other individuals and play a social role, as suggested by several kinds of evidence. Such throws were significantly more vigorous and more often used silt, rather than shells or algae, and high vigor throws were significantly more often accompanied by uniform or dark body patterns. Some throws were directed differently from beneath the arms and such throws were significantly more likely to hit other octopuses. Throws targeted at other individuals in the same population, as these appear to be, are the least common form of nonhuman throwing.

The B chromosome of Pseudococcus viburni: a selfish chromosome that exploits whole-genome meiotic drive

Vea, I. M. — 2021-08-31

Meiosis, the key process underlying sexual reproduction, is generally a fair process: each chromosome has a 50% chance of being included into each gamete. However in some organisms meiosis has become highly aberrant with some chromosomes having a higher chance of making it into gametes than others. Yet why and how such systems evolve remains unclear. Here we study the unusual reproductive genetics of mealybugs, in which only maternal-origin chromosomes are included into the gametes during male meiosis, while paternally-derived chromosomes degrade. This "whole genome meiotic drive" occurs in all males and is evolutionarily conserved. However one species - the obscure mealybug Pseudococcus viburni - has a segregating B chromosome that increases in frequency by escaping paternal genome elimination. Here we present whole-genome and gene expression data from laboratory lines with and without B chromosomes. These data allow us to identify B-linked sequences including >70 protein-coding genes as well as a B-specific satellite repeat that makes up a significant proportion of the chromosome. We also used these data to investigate the evolutionary origin of the B chromosome. The few paralogs between the B and the core genome are distributed throughout the genome, showing that it is unlikely that the B originated through a simple duplication of one of the autosomes. We also find that while many of the B-linked genes do not have paralogs within the P.viburni genome, but they do show orthology with genes in other hemipteran insects suggesting that the B might have originated from fission of one of the autosomes, possibly followed by further translocations of individual genes. Finally in order to understand the mechanisms by which the B is able to escape elimination when paternally-derived we generated gene expression data for males and females with and without B chromosomes. We find that at the developmental stage when meiosis is taking place only a small number of B-linked genes show significant expression. Only one gene was significantly over-expressed during male meiosis, which is when the drive occurs: a acetyltransferase involved in H3K56Ac, which has a putative role in meiosis and is therefore a promising candidate for further studies. Together, these results form a promising foundation for studying the mechanisms of meiotic drive in a system that is uniquely suited for this approach.

Chromosome breaks in breast cancers occur near herpes tumor virus sequences

Friedenson, B. A. — 2021-11-10

Breast cancer has a relentless tendency to come back after treatment. Analyses of public data from about 2100 breast cancers produce a model that explains this recurrence and implicates variants of Epstein-Barr viruses (EBV or Human Herpes Virus 4). These viruses cause chromosome breaks. Broken chromosome pieces rejoin abnormally, sometimes including two centromeres. Two centromeres on the same chromosome interfere with cell division. Each centromere gets pulled toward a different pole. This mechanical stress shatters chromosomes. Shattered chromosome fragments rejoin arbitrarily, but showers of mutations accompany their rejoining. In this way, a single break can destabilize the entire genome. The breast cancer phenotype is not fixed and constantly creates new cancer driver genes. The phenotype becomes independent of the original virus and its dosage. Cancer comes back because treatment does not explicitly target the underlying breakage-rejoining cycles or the contributing virus. The following data support this model. EBV causes chromosome breaks, and breast cancer chromosomes often have two centromeres. Breast cancer breakpoints on all chromosomes aggregate around the same positions as breakpoints in cancers definitively associated with EBV infection (nasopharyngeal cancer and endemic Burkitts lymphoma). Rejoined boundaries of highly fragmented chromosomes characteristic of breakage fusion cycles cluster around viral sequences. There is presumptive evidence of past infection. Human EBV sequences distribute like retrovirus transposons near dense piRNA clusters at a critical MHC-immune response region of chromosome 6. Other viruses strongly resemble endogenous transposons which piRNAs inactivate by methylation and cleavage. Remnants of exogenous EBV variants sit close to inactive transposons in piRNA sandwiches. The arrangement grossly resembles bacterial CRISPR and adds a layer of DNA protection to the immune system. Breast cancers target this protection with chromosome breaks and mutations and have a distinctive methylation signature nearby. Finally, areas near EBV docking sites can have increased numbers of breaks. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/467751v5_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@abc058org.highwire.dtl.DTLVardef@1e0ffe9org.highwire.dtl.DTLVardef@c13bc9org.highwire.dtl.DTLVardef@3c16d8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Rapid reconstruction of neural circuits using tissue expansion and lattice light sheet microscopy

Lillvis, J. L. — 2021-11-15

Electron microscopy (EM) allows for the reconstruction of dense neuronal connectomes but suffers from low throughput, limiting its application to small numbers of reference specimens. We developed a protocol and analysis pipeline using tissue expansion and lattice light-sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many samples with single synapse resolution and molecular contrast. We validate this approach in Drosophila, demonstrating that it yields synaptic counts similar to those obtained by EM, can be used to compare counts across sex and experience, and to correlate structural connectivity with functional connectivity. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

Label-free imaging of collagen fibers in tissue slices using phase imaging with computational specificity

Sakakura, M. — 2021-11-19

Evaluating the tissue collagen content in addition to the epithelial morphology has been proven to offer complementary information in histopathology, especially in oncology tumor staging and prediction of survival in cancer patients. One imaging modality widely used for this purpose is second harmonic generation microscopy (SHGM), which reports on the nonlinear susceptibility associated with the collagen fibers. Another method is polarization light microscopy (PLM) combined with picrosirius-red (PSR) tissue staining. However, SHGM requires expensive equipment and provides limited throughput, while PLM and PSR staining are not part of the routine surgical pathology workflow. Here, we utilize phase imaging with computational specificity (PICS) to computationally infer the collagen distribution of unlabeled tissue, with high specificity. PICS utilizes deep learning to translate quantitative phase images (QPI) into corresponding PSR images with high accuracy and inference speed of 200 milisecond per forwardpass through the model once trained. We developed a multimodal imaging instrument that yields both Spatial light Inference Microscopy (SLIM) and polarized light microscopy (PLM) images from the same field of view. Our results indicate that the distributions of collagen fiber orientation, length, and straightness reported by PICS closely match the ones from ground truth as defined by KL-divergence.

The protein organization of a red blood cell

Sae-Lee, W. — 2021-12-11

Red blood cells (RBCs, erythrocytes) are the simplest primary human cells, lacking nuclei and major organelles, and instead employing about a thousand proteins to dynamically control cellular function and morphology in response to physiological cues. In this study, we defined a canonical RBC proteome and interactome using quantitative mass spectrometry and machine learning. Our data reveal an RBC interactome dominated by protein homeostasis, redox biology, cytoskeletal dynamics, and carbon metabolism. We validated protein complexes through electron microscopy and chemical crosslinking, and with these data, built 3D structural models of the ankyrin/Band 3/Band 4.2 complex that bridges the spectrin cytoskeleton to the RBC membrane. The model suggests spring-link compression of ankyrin may contribute to the characteristic RBC cell shape and flexibility. Taken together, our study provides an in-depth view of the global protein organization of human RBCs and serves as a comprehensive resource for future research.

Uncovering One-Dimensional Reaction Coordinate that Underlies Structure-Function Relationship of Proteins

Wu, S. — 2022-01-10

Understanding the mechanism of functional protein dynamics is critical to understanding protein functions. Reaction coordinates is a central topic in protein dynamics and the grail is to find the one-dimensional reaction coordinate that can fully determine the value of committor (i.e. the reaction probability in configuration space) for any protein configuration. We present a powerful new method that can, for the first time, identify the rigorous one-dimensional reaction coordinate in complex molecules. This one-dimensional reaction coordinate is determined by a fundamental mechanical operator--the generalized work functional. This method only requires modest computational cost and can be readily applied to large molecules. Most importantly, the generalized work functional is the physical origin of the collectivity in functional protein dynamics and provides a tentative roadmap that connects the structure of a protein to its function.

Simultaneous dual recordings from hair cells and their calyx afferents reveal the functional significance of these specialized endings in the vestibular periphery.

Contini, D. — 2022-03-08

In the vestibular periphery, transmission via conventional synaptic boutons is supplemented by postsynaptic calyceal endings surrounding Type I hair cells. This review focusses on the multiple modes of communication between these receptors and their enveloping calyces as revealed by simultaneous dual-electrode recordings. Classic orthodromic transmission is accompanied by two forms of bidirectional communication enabled by the extensive cleft between the Type I hair cell and its calyx. The slowest cellular communication low-pass filters the transduction current with a time constant 10 -- 100 milliseconds: potassium ions accumulate in the synaptic cleft, depolarizing both the hair cell and afferent to potentials greater than necessary for rapid vesicle fusion in the receptor and potentially triggering action potentials in the afferent. On the millisecond timescale, conventional glutamatergic quantal transmission occurs when hair cells are depolarized to potentials sufficient for calcium influx and vesicle fusion. Depolarization also permits a third form of transmission which occurs over tens of microseconds, resulting from the large voltage- and ion-sensitive cleft-facing conductances in both the hair cell and the calyx that are open at their resting potentials. Current flowing out of either the hair cell or the afferent divides into the fraction flowing across the cleft into its cellular partner, and the remainder flowing out of the cleft and into the surrounding fluid compartment. These findings suggest multiple biophysical bases for the extensive repertoire of response dynamics seen in the population of primary vestibular afferent fibers. The results further suggest that evolutionary pressures drive selection for the calyx afferent.

HOIL1 regulates group 2 innate lymphoid cell numbers and type 2 inflammation in the small intestine

Wood, M. J. — 2022-03-11

Patients with mutations in HOIL1 experience a complex immune disorder including intestinal inflammation. To investigate the role of HOIL1 in regulating intestinal inflammation, we employed a mouse model of HOIL1 deficiency. The ileum of Hoil1-/- mice displayed features of type 2 inflammation including tuft cell and goblet cell hyperplasia, and elevated expression of Il13, Il5 and Il25 mRNA. Inflammation persisted in the absence of T and B cells, and bone marrow chimeric mice revealed a requirement for HOIL1 expression in radiation-resistant cells to regulate inflammation. Although disruption of IL-4 receptor alpha (IL4R) signaling on intestinal epithelial cells ameliorated tuft and goblet cell hyperplasia, expression of Il5 and Il13 mRNA remained elevated. KLRG1hi CD90lo group 2 innate lymphoid cell (ILC2) were increased independent of IL4R signaling, tuft cell hyperplasia and IL-25 induction. Antibiotic treatment dampened intestinal inflammation indicating commensal microbes as a contributing factor. We have identified a key role for HOIL1, a component of the Linear Ubiquitin Chain Assembly Complex, in regulating type 2 inflammation in the small intestine. Understanding the mechanism by which HOIL1 regulates type 2 inflammation will advance our understanding of intestinal homeostasis and inflammatory disorders and may lead to the identification of new targets for treatment.

3'UTR-directed, kinase proximal mRNA decay inhibits C/EBPβ phosphorylation/activation to suppress senescence in tumor cells

Salotti, J. — 2022-03-29

C/EBP{beta} is a potent regulator of oncogene-induced senescence (OIS) and the SASP. C/EBP{beta} is post-translationally activated in OIS cells by the effector kinases ERK1/2 and CK2. However, in tumor cells C/EBP{beta} activation is suppressed by its 3UTR. 3'UTR regulation of protein activity (UPA) requires a G/U-rich element (GRE) and its cognate binding protein, HuR. These components segregate CEBPB transcripts away from a perinuclear compartment harboring ERK1/2 and CK2, restricting C/EBP{beta} from its activating kinases. We report here that the mRNA decay proteins UPF1 and Staufen1/2 are essential UPA factors enriched within the perinuclear cytoplasm. STAU1/2 and UPF1 overlap with CK2 on perinuclear signaling endosomes where they promote localized CEBPB mRNA decay. UPF1 or STAU1/2 depletion in tumor cells increased CEBPB transcripts adjacent to CK2 foci, coinciding with C/EBP{beta} activation and senescence. The GRE and an adjacent STAU binding site independently suppress C/EBP{beta}-mediated senescence, while a distinct 3UTR region inhibits its SASP-inducing activity. KrasG12D-driven lung tumors in mice carrying a Cebpb GRE deletion rarely progressed to malignant adenocarcinomas, demonstrating the importance of UPA to enable tumor progression in vivo. Thus, kinase-proximal mRNA decay is a novel mechanism that inhibits C/EBP{beta} activation in tumor cells to facilitate senescence bypass.

Nitroimidazopyrazinones with oral activity against tuberculosis and Chagas disease in mouse models of infection

Ang, C. W. — 2022-04-06

Tuberculosis remains one of the leading causes of death from a single infectious agent, surpassing both AIDS and malaria. In recent years, two bicyclic nitroimidazole drugs, delamanid and pretomanid have been approved to treat this airborne infection. This has spurred a renewed interest in developing new and improved nitroimidazole analogs. We have previously identified a new bicyclic heteroaromatic subclass, the nitroimidazopyrazinones, with substituted analogs showing promising activity against Mycobacterium tuberculosis under both aerobic and hypoxic environments. A second generation of nitroimidazopyrazinones with extended biaryl side chain also possessed good antiparasitic activity against Trypanosoma brucei brucei and Trypanosoma cruzi, suggesting the utility of this new scaffold for development into potential candidates against both tuberculosis and the kinetoplastid parasites which cause neglected tropical diseases. In this study, we further evaluated the properties of nitroimidazopyrazinone derivatives by assessing their selectivity against different mycobacterial species, measuring their reduction potential, and determining the kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn), which is the activating enzyme of delamanid and pretomanid in M. tuberculosis. We also conducted an in vivo evaluation of a lead compound, MCC8967 that demonstrated a favorable pharmacokinetic profile, with good oral bioavailability and efficacy in an acute M. tuberculosis infection model. Two other promising compounds MCC9481 and MCC9482, with good in vitro activity (IC50 = 0.016 and 0.10 {micro}M, respectively) against T. cruzi, the causative agent for Chagas diseases, were similarly tested for in vivo activity. These compounds also exhibited good oral bioavailability, and transiently reduced the acute-stage parasite burden by >98-99% at doses of 50 mg/kg once or twice daily, similar to benznidazole at 100 mg/kg once daily. Overall, we have demonstrated that active nitroimidazopyrazinones have potential to be developed as clinical candidates against both tuberculosis and Chagas disease. Author SummaryTuberculosis and parasitic infections continue to impose a significant threat to public health and economic growth worldwide. Most of the efforts to control these diseases still rely on drug treatments with limited effectiveness and significant side effects. There is now an urgent need to develop new treatments to combat these infections. Here, we report the in vitro and in vivo profile of a new bicyclic nitroimidazole subclass, namely nitroimidazopyrazinones, against mycobacteria and Trypanosoma cruzi. We found that derivatives with monocyclic side chains are selective against Mycobacterium tuberculosis, the causative agent of tuberculosis, but not active against other nontuberculosis mycobacteria. In an acute mouse model, they were able to reduce the bacterial load in lungs via oral administration. From a biochemistry perspective, we demonstrated that deazaflavin-dependent nitroreductase (Ddn) could act effectively on nitroimidazopyrazinones, indicating the potential of Ddn as an activating enzyme for these new compounds in M. tuberculosis. We also showed that derivatives with extended biaryl side chain were effective in suppressing infection in an acute T. cruzi infected murine model, with satisfactory oral bioavailability. These findings improve the understanding of the biological profile of nitroimidazopyrazinones for further development as potential antitubercular and antiparasitic agents.

Origin of Protein Quake: Energy Waves Conducted by a Precise Mechanical Machine

Li, H. — 2022-04-13

A long-standing challenge in protein biophysics is to understand protein quake in myoglobin--the structural dynamics responsible for redistributing the excess heme energy after photolysis. Despite extensive efforts, the molecular mechanism of this process remains elusive. Using the energy flow theory, we uncovered a fundamental new phenomenon: the heme energy is redistributed by energy waves with a ubiquitous fundamental frequency and two overtones. Energy waves emanate from the heme into the myoglobin backbone via a conduit of five consecutive dihedrals of the proximal histidine, then travel quickly along the backbone to reach sidechains across the protein. This mechanism is far more effective than the diffusion-based mechanism from previous studies because waves are systematic while diffusion is random. To propagate energy waves, coordinates must cooperate, resulting in collective modes that are singular vectors of the generalized work functional. These modes show task partitioning: a handful of high-energy modes generate large-scale breathing motion, which loosens up the protein matrix to enable hundreds of low-energy vibrational modes for energy transduction.

Probing the force-from-lipid mechanism with synthetic polymers

Jacobs, M. L. — 2022-05-21

A central feature of mechanotransduction is the ability of mechanosensitive channels to respond to mechanical stimuli from the surrounding lipid bilayer. Accordingly, the mechanical properties of membranes should play an important role in modulating force transmission to embedded channels, yet the nature of this relationship remains unclear for a wide class of mechanosensitive channels across prokaryotic and eukaryotic systems. Here, we use a synthetic amphiphile to modulate the membrane mechanical properties of cell-derived vesicles and probe channel activation. Using precise membrane mechanical characterization approaches that have rarely been used in conjunction with electrophysiology techniques, we directly characterize three membrane properties and the activation threshold of the E. coli mechanosensitive channel of large conductance (MscL). Our study reveals that decreases in the membrane area expansion modulus, KA, and bending rigidity, kc, correlate with increases in the pressure required to activate MscL and that this effect is reproducible with the mammalian channel, TREK-1. MD simulations demonstrate that polymer-mediated changes in interfacial tension is the best mechanism to describe these experimental results. Together, our results bolster the force-from-lipids mechanism by demonstrating the generality of the relationship between changes in specific membrane mechanical properties and the gating pressure of MscL and TREK-1. In addition, our results reveal the mechanical mechanism by which membrane amphiphiles alter the activity and sensitivity of mechanosensitive channels through changes in long-range force transmission.

The Genetic Architecture of Morphological Scaling

Wilcox, A. S. — 2022-06-07

Morphological scaling relationships between the sizes of individual traits and the body captures the characteristic shape of a species, and the evolution of scaling is the primary mechanism of morphological diversification. However, we have almost no knowledge of the genetic architecture of scaling, critical if we are to understand how scaling evolves. Here we explore the genetic architecture of population-level morphological scaling relationships - the scaling relationship fit to multiple genetically-distinct individuals in a population - by describing the distribution of individual scaling relationships - genotype-specific scaling relationships that are unseen or cryptic. These individual scaling relationships harbor the genetic variation that determines relative trait growth within individuals, and theoretical studies suggest that their distribution dictates how the population scaling relationship will respond to selection. Using variation in nutrition to generate size variation within 197 isogenic lineages of Drosophila melanogaster, we reveal extensive variation in the slopes of the wing-body and leg-body scaling relationships among individual genotypes. This genetic variation reflects variation in the nutritionally-induced size plasticity of the wing, leg and body. Surprisingly, we find that variation in the slope of individual scaling relationships primarily results from variation in nutritionally-induced plasticity of body size, not leg or wing size. These data allow us to predict how different selection regimes alter scaling in Drosophila and is the first step in identifying the genetic targets of such selection. More generally, our approach provides a framework for understanding the genetic architecture of scaling, an important prerequisite to explaining how selection changes scaling and morphology.

Avian and Human Influenza Viruses Exhibit Distinct Glycoconjugate Receptor Specificities in Human Lung Cells

Liang, C.-Y. — 2022-06-30

IAV utilize sialic acid (Sia) containing cell surface glycoconjugates for host cell infection, and IAV strains from different host species show preferences for structurally distinct Sia at the termini of glycoconjugates. Various types of cell surface glycoconjugates (N-glycans, O-glycans, glycolipids) display significant diversity in both structure and carbohydrate composition. To define the types of glycoconjugates that facilitate IAV infection, we utilized the CRISPR/Cas9 technique to truncate different types of glycoconjugates, either individually or in combination, by targeting glycosyltransferases essential to glycan biosynthesis in a human lung epithelial cell line. Our studies show that both human and avian IAV strains do not display strict preferences for a specific type of glycoconjugate. Interestingly, truncation of all three types of glycoconjugates significantly decreased the replication of human IAV strains, yet did not impact the replication of avian IAV strains. Taken together, our studies demonstrate that avian IAV strains utilize a broader repertoire of glycoconjugates for host cell infection as compared to human IAV strains. Author SummaryIt is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These glycoconjugates can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugates for host cell infection. Here, we utilized CRISPR gene editing to abolish sialic acid on different glycoconjugate types in human lung cells, and evaluated human versus avian IAV infections. Our studies show that both human and avian IAV strains can infect human lung cells by utilizing any of the three major sialic acid-containing glycoconjugate types, specifically N-glycans, O-glycans, and glycolipids. Interestingly, simultaneous elimination of sialic acid on all three glycoconjugate types in human lung cells dramatically decreased human IAV infection, yet had little effect on avian IAV infection. Our studies indicate that avian IAV strains can utilize a wide variety of glycoconjugates for infection, whereas human IAV strains display restrictions in glycoconjugate type usage. These novel studies show distinct differences in host glycoconjugate preferences between human and avian IAV strains.

Transcriptional changes in human palate and skin healing

Leonardo, T. R. — 2022-09-03

Most human tissue injuries lead to the formation of a fibrous scar and result in the loss of functional tissue. One adult tissue that exhibits a more regenerative response to injury with minimal scarring is the oral mucosa. We generated a microarray gene expression dataset to examine the response to injury from human palate and skin excisional biopsies spanning the first seven days after wounding. Differential expression analyses were performed in each tissue to identify genes overexpressed or underexpressed over time compared to baseline unwounded tissue. To attribute biological processes of interest to these gene expression changes, gene set enrichment analysis was used to identify core gene sets that are enriched over the time-course of the wound healing process with respect to unwounded tissue. This analysis identified gene sets uniquely enriched in either palate or skin wounds and gene sets that are enriched in both tissues in at least one time point after injury. Finally, a cell-deconvolution analysis was performed to better understand the cell type distribution in these tissues and how it changes over the time course of wound healing. This work provides a source of human wound gene expression data that includes two tissue types with distinct regenerative and scarring phenotypes.

Self-assembly of cellular neighborhoods converts stochastic signaling into sustained olfactory neurogenesis

Rajan, S. G. — 2022-09-06

Olfactory neurogenesis occurs continuously throughout the lives of vertebrates, including in humans, and relies on the rapid, unceasing differentiation and integration of neurons into a complex multicellular network. The system-wide regulation of this intricate choreography is poorly understood; in particular, it is unclear how progenitor cells convert stochastic fluctuations in cell-cell signaling, over both space and time, into streamlined fate decisions. Here, we track single-cell level multicellular dynamics in the developing zebrafish olfactory epithelium, perturb signaling pathways with temporal specificity, and find that the continuous generation of neurons is driven by the spatially-restricted self-assembly of transient groups of progenitor cells, i.e. cellular neighborhoods. Stochastic modeling and validation of the underlying genetic circuit reveals that neighborhood self-assembly is driven by a tightly regulated bistable toggle switch between Notch signaling and the transcription factor Insulinoma-associated 1a that is responsive to inter-organ retinoic acid signaling. Newly differentiating neurons emerge from neighborhoods and, in response to brain-derived neurotrophic factor signaling, migrate across the olfactory epithelium to take up residence as apically-located, mature sensory neurons. After developmental olfactory neurogenesis is complete, inducing injury results in a robust expansion of neighborhoods, followed by neuroregeneration. Taken together, these findings provide new insights into how stochastic signaling networks spatially pattern and regulate a delicate balance between progenitors and their neuronal derivatives to drive sustained neurogenesis during both development and regeneration.

New Structure of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis suggest a novel catalytic mechanism for the entire class

Abad-Zapatero, C. — 2022-09-06

Class II Fructose-1,6-bisphosphatases (FBPaseII) (EC: 3.1.3.11) are highly conserved essential enzymes in the gluconeogenic pathway of microorganisms. Previous crystallographic studies of FBPasesII provided insights into various inactivated states of enzymes from different species. Presented here is the first crystal structure of FBPaseII in an active state, solved for the enzyme from Francisella tularensis (FtFBPaseII), containing native metal cofactor Mn2+ and complexed with catalytic product fructose-6-phosphate (F6P). Another crystal structure of the same enzyme complex is presented in inactivated state due to the structural changes introduced by crystal packing. Analysis of the interatomic distances among the substrate, product and divalent metal cations in the enzyme catalytic centers led to a revision of the catalytic mechanism suggested previously for class II FBPases. Instead of a metal cofactor for the stabilization of the transition state of the leaving phosphate group, we propose that the positive dipole of the neighboring -helix backbone (G88-T89-T90-I91-T92-S93-K94) is responsible for retaining the cleaved phosphate. The revised catalytic mechanism involves a nucleophilic attack by a reactive water coordinated by both the T89 hydroxyl and a second water molecule coordinated directly by Mn2+. Additionally, a crystal structure of Mycobacterium tuberculosis FBPaseII (MtFBPaseII), containing the substrate fructose-1,6-bisphosphate (F1,6BP) bound, is presented in support of a novel catalytic mechanism for this class of enzymes.

Genetic Regulators of Sputum Mucin Concentration and Their Associations with COPD Phenotypes

Van Buren, E. — 2022-09-28

Hyper-secretion and/or hyper-concentration of mucus is a defining feature of multiple obstructive lung diseases, including chronic obstructive pulmonary disease (COPD). Mucus itself is composed of a mixture of water, ions, salt and proteins, of which the gel-forming mucins, MUC5AC and MUC5B, are the most abundant. Recent studies have linked the concentrations of these proteins in sputum to COPD phenotypes, including chronic bronchitis (CB) and acute exacerbations (AE). We sought to determine whether common genetic variants influence sputum mucin concentrations and whether these variants are also associated with COPD phenotypes, specifically CB and AE. We performed a GWAS to identify quantitative trait loci for sputum mucin protein concentration (pQTL) in the Sub-Populations and InteRmediate Outcome Measures in COPD Study (SPIROMICS, n=708 for total mucin, n=215 for MUC5AC, MUC5B). Subsequently, we tested for associations of mucin pQTL with CB and AE using regression modeling (n=822-1300). Replication analysis was conducted using data from COPDGene (n =5740) and by examining results from the UK Biobank. We identified one genome-wide significant pQTL for MUC5AC (rs75401036) and two for MUC5B (rs140324259, rs10001928). The strongest association for MUC5B, with rs140324259 on chromosome 11, explained 14% of variation in sputum MUC5B. Despite being associated with lower MUC5B, the C allele of rs140324259 conferred increased risk of CB (odds ratio (OR) = 1.42; 95% confidence interval (CI): 1.10-1.80) as well as AE ascertained over three years of follow up (OR=1.41; 95% CI: 1.02-1.94). Associations between rs140324259 and CB or AE did not replicate in COPDGene. However, in the UK Biobank, rs140324259 was associated with phenotypes that define CB, namely chronic mucus production and cough, again with the C allele conferring increased risk. We conclude that sputum MUC5AC and MUC5B concentrations are associated with common genetic variants, and the top locus for MUC5B may influence COPD phenotypes, in particular CB. Author SummaryChronic obstructive pulmonary disease (COPD) is characterized by presence of emphysema and/or chronic bronchitis. Excessive mucus production is a defining phenotype of chronic bronchitis, and is associated with several important features of COPD, including exacerbations and loss of lung function. Recent studies have demonstrated that the amount of mucus produced in COPD patients is an important marker of disease state. We investigated whether common genetic variants are associated with the concentration of two key proteins in mucus, MUC5AC and MUC5B, and whether the variants we identified are also associated with COPD outcomes. We identified multiple genetic variants that were associated with MUC5AC or MUC5B concentration. The strongest association we detected, for MUC5B on chromosome 11, was also associated with features of COPD, including chronic bronchitis and acute exacerbations, in one COPD study population but not another. Results from a much larger study, the UK Biobank, indicate that this variant is associated with chronic mucus production and chronic cough, which are key features of chronic bronchitis. Thus, we conclude that the concentration of key proteins in mucus are influenced by genetic variation, and that a variant on chromosome 11 that affects MUC5B may in turn alter COPD outcomes.

Genomic Analysis of Two Phlebotomine Sand Fly Vectors of Leishmania from the New and Old World

McDowell, M. A. — 2022-10-05

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the devastating kinetoplastid parasites of the genus Leishmania, the causative agents of diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. As no approved efficacious vaccine exists, available drugs are expensive and/or toxic, and resistance is emerging, management of sand fly populations to break transmission is currently the most effective disease control strategy. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two of the most important sand fly species: Phlebotomus papatasi, a cutaneous leishmaniasis vector, (distributed in the Middle East and North Africa) and Lutzomyia longipalpis, a visceral leishmaniasis vector (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites. Author SummaryThe leishmaniases are a group of neglected tropical diseases caused by protist parasites from the Genus Leishmania. Different Leishmania species present a wide clinical profile, ranging from mild, often self-resolving cutaneous lesions that can lead to protective immunity, to severe metastatic mucosal disease, to visceral disease that is ultimately fatal. Leishmania parasites are transmitted by the bites of sand flies, and as no approved vaccine exists, available drugs are toxic and/or expensive and resistance is emerging, new dual control strategies to combat these diseases must be developed, combining interventions on human infections and integrated sand fly population management. Effective vector control requires a good understanding of the biology of sand flies. To this end, we sequenced and annotated the genomes of two sand fly species that are important leishmaniasis vectors from the Old and New Worlds. These genomes allow us to better understand, at the genetic level, processes important in the vector biology of these species, such as finding hosts, blood-feeding, immunity, and detoxification. These genomic resources highlight the driving forces of evolution of two major Leishmania vectors and provide foundations for future research on how to better prevent leishmaniasis by control of the sand fly vectors.

Impaired Barrier Integrity of the Skeletal Muscle Vascular Endothelium Drives Progression of Cancer Cachexia

Kim, Y.-M. — 2022-12-14

Cancer patients experience cachexia, which is characterized by extensive skeletal muscle wasting that worsens the quality of life and increases mortality. Currently, there are no approved treatments that can effectively counteract cancer cachexia. Vascular endothelial cells (ECs) are essential for maintaining tissue perfusion, nutrient supply, and preventing inappropriate transmigration of immune cells into the tissue. However, little is known about the role of the muscle vasculature in cancer cachexia. We hypothesized that endothelial dysfunction in the skeletal muscle mediates cancer cachexia. Using transgenic pancreatic ductal adenocarcinoma (PDAC) mice and a tissue clearing and high-resolution 3D-tissue imaging approach, we found that the loss of skeletal muscle vascular density precedes the loss of muscle mass. Importantly, we show that cancer cachexia patients exhibit significantly decreased muscle vascular density and severe muscle atrophy when compared to non-cancer patients. Unbiased single cell transcriptomic analyses of the muscle endothelium unveiled a unique EC population present in cachexia muscles. Increased circulating Activin-A suppresses the expression of the transcriptional co-activator PGC1 in the muscle endothelium, thus disrupting junctional integrity in the vasculature and increasing vascular leakage. Conversely, restoration of endothelial-specific PGC1 prevented the decreased vascular density and muscle loss observed in tumor-bearing mice. Our study suggests that EC-PGC1 is essential for maintaining the integrity of the skeletal muscle vascular barrier and that restoring muscle endothelial function could be a valuable therapeutic approach to prevent or reverse cancer cachexia.

Cdkn2a regulates beige fat maintenance through BECN1-mediated autophagy

Wu, R. — 2022-12-22

A potential therapeutic target to curb the obesity and diabetes epidemic is thermogenic beige adipocytes. However, beige adipocytes quickly transition into white adipocytes upon removing stimuli. Here, we define the critical role of Cdkn2a as a molecular pedal for the beige-to-white transition. Beige adipocytes lacking Cdkn2a exhibit prolonged lifespan, and mice are more resistant to diet-induced obesity, along with enhanced energy expenditure and improved glucose tolerance. Mechanistic studies demonstrate that Cdkn2a promotes the expression and activity of BECN1 by directly binding to its mRNA and its negative regulator BCL2L1, activating autophagy and accelerating the beige-to-white transition. Notably, reactivating autophagy by pharmacological or genetic methods abolishes beige adipocyte maintenance induced by Cdkn2a-ablation. Furthermore, hyperactive BECN1 alone significantly accelerates the beige-to-white transition. Collectively, these findings show that Cdkn2a-mediated autophagy serves as a brake system for beige adipocyte maintenance and is a highly promising target for anti-obesity and anti-diabetes therapy. HighlightsO_LICdkn2a ablation promotes beige fat maintenance and ameliorates diet-induced obesity C_LIO_LILoss of Cdkn2a retains beige adipocytes by inhibiting BECN1-mediated autophagy C_LIO_LICdkn2a modulates BECN1 by binding to its mRNA and its inhibitor BCL2L1, respectively C_LIO_LIHyperactive BECN1 is sufficient to accelerate the beige-to-white transition C_LI

DBDA matrix increases ion abundance of fatty acids and sulfatides in MALDI-TOF and mass spectrometry imaging studies

Khamidova, N. — 2023-01-14

MALDI-TOF MS is a powerful tool to analyze biomolecules owing to its soft ionization nature and generally results in simple spectra of singly charged ions. Moreover, implementation of the technology in imaging mode provides a means to spatially map analytes in situ. Recently, a new matrix, DBDA (N1,N4-dibenzylidenebenzene-1,4-diamine) was reported to facilitate the ionization of free fatty acids in the negative ion mode. Building on this finding, we sought to implement DBDA for MALDI mass spectrometry imaging studies in brain tissue and successfully map oleic acid, palmitic acid, stearic acid, docosahexaenoic acid and arachidonic acid using mouse brain sections. Moreover, we hypothesized that DBDA would provide superior ionization for sulfatides, a class of sulfolipids, with multiple biological functions. Herein we also demonstrate that DBDA is ideal for MALDI mass spectrometry imaging of fatty acids and sulfatides in brain tissue sections. Additionally, we show enhanced ionization of sulfatides using DBDA compared to three different traditionally used MALDI matrices. Together these results provide new opportunities for studies to measure sulfatides by MALDI-TOF MS including in imaging modes.

PI(3,5)P2 Controls the Signaling Activity of Class I PI3K

Sun, J. — 2023-01-25

3-Phosphoinositides are ubiquitous cellular lipids that play pivotal regulatory roles in health and disease. Among 3-phosphoinositides, phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) remains the least understood species in terms of its spatiotemporal dynamics and physiological function due to the lack of a specific sensor that allows spatiotemporally resolved quantitative imaging of PI(3,5)P2. Using a newly developed ratiometric PI(3,5)P2 sensor engineered from the C-terminal SH2 domain of Class I phosphoinositide 3-kinases (PI3K)-p85 subunit we demonstrate that a unique pool of PI(3,5)P2 is generated on lysosomes and late endosomes in response to growth factor stimulation. This PI(3,5)P2, the formation of which is mediated sequentially by Class II PI3KC2{beta} and PIKfyve, plays a crucial role in terminating the activity of growth factor-stimulated Class I PI3K, one of the most frequently mutated proteins in cancer, via specific interaction with its regulatory p85 subunit. A small molecule inhibitor of p85-PI(3,5)P2 binding specifically blocks the feedback inhibition of Class I PI3K by PI(3,5)P2 and thus serves as a PI3K activator that promotes neurite growth. Furthermore, cancer-causing mutations of the Class I PI3K-p85 subunit inhibit p85-PI(3,5)P2 interaction and thereby induce sustained activation of Class I PI3K. Our results unravel a hitherto unknown spatiotemporally specific regulatory function of PI(3,5)P2 that links Class I and II PI3Ks and modulates the magnitude of PI3K-mediated growth factor signaling. These results also suggest new therapeutic possibilities for treating cancer patients with p85 mutations and promoting wound healing and tissue regeneration.

Homeostatic Reinforcement Theory Accounts for Sodium Appetitive State- and Taste- Dependent Dopamine Responding.

Duriez, A. — 2023-01-27

Seeking and consuming nutrients is essential to survival and maintenance of life. Dynamic and volatile environments require that animals learn complex behavioral strategies to obtain the necessary nutritive substances. While this has been classically viewed in terms of homeostatic regulation, where complex nutrient seeking behaviors are triggered by physiological need, recent theoretical work proposed that such strategies are a result of reinforcement learning processes. This theory also proposed that phasic dopamine (DA) signals play a key role in signaling potentially need-fulfilling outcomes. To examine potential links between homeostatic and reinforcement learning processes, we focus on sodium appetite as sodium depletion triggers state and taste dependent changes in behavior and DA signaling evoked by sodium-related stimuli. We find that both the behavior and the dynamics of DA signaling underlying sodium appetite can be accounted for by extending principles of homeostatic regulation into a reinforcement learning framework (HRRL). We first optimized HRRL-based agents to model sodium-seeking behavior measured in rats. Agents successfully reproduced the state and the taste dependence of behavioral responding for sodium as well as for lithium and potassium salts. We then show that these same agents can account for the regulation of DA signals evoked by sodium tastants in a taste and state dependent manner. Our models quantitatively describe how DA signals evoked by sodium decrease with satiety and increase with deprivation suggesting that phasic DA signals and sodium consumption are down regulated prior to animals reaching satiety. Lastly, our HRRL agents also account for the behavioral and neurophysiological observations that suggest mice cannot distinguish between sodium and lithium containing salts. Our HRRL agents exhibited an equal preference for sodium versus lithium containing solutions, and underestimated the nutritional value of sodium when lithium was concurrently available. We propose that animals use orosensory signals as predictors of the internal impact of the consumed good and our results pose clear targets for future experiments. In sum, this work suggests that appetite-dirven behavior may be driven by reinforcement learning mechanisms that are dynamically tuned by homeostatic need.

Three-dimensional spatial quantitative analysis of cardiac lymphatics in the mouse heart

Phillips, E. H. — 2023-02-03

Objective3D microscopy and image data analysis are necessary for studying the morphology of cardiac lymphatic vessels (LyVs) and association with other cell types. We aimed to develop a methodology for 3D multiplexed lightsheet microscopy and highly sensitive and quantitative image analysis to identify pathological remodeling in the 3D morphology of LyVs in young adult mouse hearts with familial hypertrophic cardiomyopathy (HCM). MethodsWe developed a 3D lightsheet microscopy workflow providing a quick turn-around (as few as 5-6 days), multiplex fluorescence detection, and preservation of LyV structure and epitope markers. Hearts from non-transgenic (NTG) and transgenic (TG) HCM mice were arrested in diastole, retrograde perfused, immunolabeled, optically cleared, and imaged. We built an image processing pipeline to quantify LyV morphological parameters at the chamber and branch levels. ResultsChamber-specific pathological alterations of LyVs were identified, but most significantly in the right atrium (RA). TG hearts had a higher volume fraction of ER-TR7+ fibroblasts and reticular fibers. In the RA, we found associations between ER-TR7+ volume fraction and both LyV segment density and median diameter. ConclusionsThis workflow and study enabled multi-scale analysis of pathological changes in cardiac LyVs of young adult mice, inviting ideas for research on LyVs in cardiac disease.

LncRNA MALAT1/microRNA-30b axis regulate macrophage polarization and function

Ahmad, I. — 2023-02-03

IntroductionMacrophages (M{varphi}) can polarize towards the proinflammatory M1 or proresolving M2 phenotype to control diverse biological processes such as inflammation, and tissue regeneration. Noncoding RNAs play critical roles in numerous biological pathways; however, their functional interaction in the regulation of M{varphi} polarization and immune responses remain unclear. ObjectivesTo examine relationship between lncRNA (MALAT1) and microRNA (miR-30b) in shaping macrophage polarization and immune functions. MethodsExpression of MALAT1 and miR-30b was examined in differentiating M1/M2 M{varphi}, human and murine inflamed gingival biopsies by RT-qPCR. MALAT1 and miR-30b direct interaction was examined by dual luciferase assays. Impact of MALAT1 knockdown and miR-30b overexpression was examined on macrophage polarization markers, bacterial phagocytosis, antigen uptake/processing and cytokine profiles. ResultsMALAT1 expression displays a time-dependent induction during M{varphi} differentiation and, upon challenge with TLR4 agonist (E. coli LPS). Knockdown of MALAT1 enhanced the expression of M2M{varphi} markers without affecting the M1M{varphi} markers, suggesting that MALAT1 favors the M1 phenotype by suppressing M2 polarization. MALAT1 knockdown M{varphi} exhibit reduced antigen uptake and processing, bacterial phagocytosis, and bactericidal activity, strongly supporting its critical role in regulating innate immune functions. Consistent with this, MALAT1 knockdown showed impaired cytokine secretion upon challenge with LPS. Importantly, MALAT1 exhibit an antagonistic expression pattern with all five members of the miR-30 family during M2M{varphi} differentiation. Dual-luciferase assays validated a novel sequence on MALAT1 that interacts with miR-30b, a microRNA that promotes the M2 phenotype. Phagocytosis and antigen processing assays unequivocally demonstrated that MALAT1 and miR-30b are functionally antagonistic. In human subjects with periodontal disease and murine model of ligature-induced periodontitis, we observed higher levels of MALAT1, and downregulation of miR-30b that correlates with higher M1M{varphi} markers expression in gingival tissues suggesting a pro-inflammatory function of MALAT1. ConclusionMALAT1/miR-30b antagonistic interaction shapes M{varphi} polarization in vitro and in inflamed gingival biopsies.

Solid Phase Synthesis of Fluorosulfate Containing Macrocycles for Chemoproteomic Workflows

Faucher, F. F. — 2023-02-18

Macrocyclic peptides are attractive for chemoproteomic applications due to their modular synthesis and potential for high target selectivity. We describe a solid phase synthesis method for the efficient generation of libraries of small macrocycles that contain an electrophile and alkyne handle. The modular synthesis produces libraries that can be directly screened using simple SDS-PAGE readouts and then optimal lead molecules applied to proteomic analysis. We generated a library of 480 macrocyclic peptides containing the weakly reactive fluorosulfate (OSF) electrophile. Initial screening of a subset of the library containing each of the various diversity elements identified initial molecules of interest. The corresponding positional and confirmational isomers were then screened to select molecules that showed specific protein labeling patterns that were dependent on the probe structure. The most promising hits were applied to standard chemoproteomic workflows to identify protein targets. Our results demonstrate the feasibility of rapid, on-resin synthesis of diverse macrocyclic electrophiles to generate new classes of covalent ligands.

Dynamic Changes in Macrophage Polarization during the Resolution Phase of Periodontal Disease

Uttamani, J. — 2023-02-21

Periodontal inflammation is largely governed by infiltration of myeloid cells, in particular macrophages. Polarization of M{varphi} within the gingival tissues is a well-controlled axis and has considerable consequences for how M{varphi} participate in inflammatory and resolution (tissue repair) phases. We hypothesize that periodontal therapy may instigate a pro-resolution environment favoring M2 M{varphi} polarization and contribute towards resolution of inflammation post-therapy. We aimed to evaluate the markers of macrophage polarization before and after periodontal therapy. Gingival biopsies were excised from human subjects with generalized severe periodontitis, undergoing routine non-surgical therapy. A second set of biopsies were excised after 4-6 weeks to assess the impact of therapeutic resolution at the molecular level. As controls, gingival biopsies were excised from periodontally healthy subjects, undergoing crown lengthening. Total RNA was isolated from gingival biopsies to evaluate pro- and anti-inflammatory markers associated with macrophage polarization by RT-qPCR. Mean periodontal probing depths, CAL and BOP reduced significantly after therapy and corroborated with the reduced levels of periopathic bacterial transcripts after therapy. Compared to heathy and treated biopsies, higher load of Aa and Pg transcripts were observed in disease. Lower expression of M1M{varphi} markers (TNF-, STAT1) were observed after therapy as compared to diseased samples. Conversely, M2M{varphi} markers (STAT6, IL-10) were highly expressed in post-therapy as opposed to pre-therapy, which correlated with clinical improvement. These findings corroborated with murine ligature-induced periodontitis and resolution model, comparing the respective murine M{varphi} polarization markers (M1 M{varphi}: cox2, iNOS2 and M2 M{varphi}: tgm2 and arg1). Our findings suggest that imbalance in M1 and M2 polarized macrophages by assessment of their markers can provide relevant clinical information on the successful response of periodontal therapy and can be used to target non-responders with exaggerated immune responses.

Long noncoding RNA AL109754.1 Regulates Myeloid Dendritic Cell Differentiation and Potentiates TLR signaling

Naqvi, R. — 2023-03-02

Dendritic cells (DCs) are key antigen presentation cells (APC) that bridge innate and adaptive immune functions to contain the pathogenic threats. Long noncoding RNAs (lncRNAs) are implicated in functional regulation of various biological processes including inflammation and immunity. However, the knowledge on myeloid DC expressed lncRNA repertoire and their regulatory functions is limited. In this study, we have reconnoitered the time-kinetics of lncRNA expression profiles during monocyte-to-DC differentiation and their roles in shaping DC functions. Our RNA-seq data identified thousands of differentially expressed lncRNAs associated with primary human monocyte-to-DC differentiation in vitro. We selected two lncRNAs viz., AL109754.1 and AC093278.2 that were enriched during DC differentiation. Knockdown of AL109754.1 but not AC093278 affects DCs differentiation as observed by marked reduction of surface markers CD1a, CD93 and CD209. These DCs also exhibit significant reduction in the expression of TLR 2, 4, 5, 7 and 9, suggesting that AL109754.1 expression is critical in maintaining TLR expression in DCs. Furthermore, reduced phosphorylation of NF-{kappa}B, IRF3 and IRF7 in AL109754.1 knockdown DCs treated with TLR agonists further substantiate their role in potentiating TLR signaling. Mechanistically, AL109754.1 knockdown DC showed significant downregulation of multiple NF-{kappa}B-induced genes and time-dependent inhibition of pro-inflammatory cytokine (IL-1{beta}, IL-6, IL-8 and TNF) secretion upon challenge with TLR 4, 5, or 7 agonists. Overall, this study characterized novel functions of AL109754.1 that regulates DC differentiation, and TLR-dependent innate immune activation.

Comprehensive mutational analysis of the checkpoint signaling functions of Rpa1/Ssb1 in fission yeast

Xu, Y. — 2023-03-06

Replication protein A (RPA) is a heterotrimeric complex and the major single-strand DNA (ssDNA) binding protein in eukaryotes. It plays important roles in DNA replication, repair, recombination, telomere maintenance, and checkpoint signaling. Because RPA is essential for cell survival, understanding its checkpoint signaling function in cells has been challenging. Several RPA mutants have been reported previously in fission yeast. None of them, however, has a defined checkpoint defect. A separation-of-function mutant of RPA, if identified, would provide significant insights into the checkpoint initiation mechanisms. We have explored this possibility and carried out an extensive genetic screening for Rpa1/Ssb1, the large subunit of RPA in fission yeast, looking for mutants with defects in checkpoint signaling. This screen has identified twenty-five primary mutants that are sensitive to genotoxins. Among these mutants, two have been confirmed partially defective in checkpoint signaling primarily at the replication fork, not the DNA damage site. The remaining mutants are likely defective in other functions such as DNA repair or telomere maintenance. Our screened mutants, therefore, provide a valuable tool for future dissection of the multiple functions of RPA in fission yeast. AUTHOR SUMMARYOriginally discovered as a protein required for replication of simian virus SV40 DNA, replication protein A is now known to function in DNA replication, repair, recombination, telomere maintenance, and checkpoint signaling in all eukaryotes. The protein is a complex of three subunits and the two larger ones are essential for cell growth. This essential function however complicates the studies in living cells, and for this reason, its checkpoint function remains to be fully understood. We have carried out an genetic screening of the largest subunit of this protein in fission yeast, aiming to find a non-lethal mutant that lacks the checkpoint function. This extensive screen has uncovered two mutants with a partial defect in checkpoint signaling when DNA replication is arrested. Surprisingly, although the two mutants also have a defect in DNA repair, their checkpoint signaling remains largely functional in the presence of DNA damage. We have also uncovered twenty-three mutants with defects in DNA repair or telomere maintenance, but not checkpoint signaling. Therefore, the non-lethal mutants uncovered by this study provide a valuable tool for dissecting the multiple functions of this biologically important protein in fission yeast.

The ComRS-SigX pathway regulates natural transformation in Streptococcus ferus

Rued, B. E. — 2023-03-07

The ability to take up and incorporate foreign DNA via natural transformation is a well-known characteristic of some species of Streptococcus, and is a mechanism that rapidly allows for the acquisition of antibacterial resistance. Here, we describe that the understudied species Streptococcus ferus is also capable of natural transformation and uses a system analogous to that identified in Streptococcus mutans. S. mutans natural transformation is under the control of the alternative sigma factor sigX (also known as comX), whose expression is induced by two types of peptide signals: CSP (competence stimulating peptide, encoded by comC) and XIP (sigX-inducing peptide, encoded by comS). These systems induce competence via either the two-component signal-transduction system ComDE or the RRNPP transcriptional regulator ComR, respectively. Protein and nucleotide homology searches identified putative orthologs of comRS and sigX in S. ferus, but not homologs of S. mutans blpRH (also known as comDE). We demonstrate that natural transformation in S. ferus is induced by a small, double-tryptophan containing competence-inducing peptide (XIP), akin to that of S. mutans, and requires the presence of the comR and sigX orthologs for efficient transformation. Additionally, we find that natural transformation is induced in S. ferus by both the native XIP and the XIP variant of S. mutans, implying that crosstalk between the two species is possible. This process has been harnessed to construct gene deletions in S. ferus and provides a method to genetically manipulate this understudied species. IMPORTANCENatural transformation is the process by which bacteria take up DNA and allows for acquisition of new genetic traits, including those involved in antibiotic resistance. This study demonstrates that the understudied species Streptococcus ferus is capable of natural transformation using a peptide-pheromone system like that previously identified in Streptococcus mutans and provides a framework for future studies concerning this organism.

Manipulation of the nucleoscaffold potentiates cellular reprogramming kinetics

Yang, B. A. — 2023-03-13

Somatic cell fate is an outcome set by the activities of specific transcription factors and the chromatin landscape and is maintained by gene silencing of alternate cell fates through physical interactions with the nuclear scaffold. Here, we evaluate the role of the nuclear scaffold as a guardian of cell fate in human fibroblasts by comparing the effects of transient loss (knockdown) and mutation (progeria) of functional Lamin A/C, a core component of the nuclear scaffold. We observed that Lamin A/C deficiency or mutation disrupts nuclear morphology, heterochromatin levels, and increases access to DNA in lamina-associated domains. Changes in Lamin A/C were also found to impact the mechanical properties of the nucleus when measured by a microfluidic cellular squeezing device. We also show that transient loss of Lamin A/C accelerates the kinetics of cellular reprogramming to pluripotency through opening of previously silenced heterochromatin domains while genetic mutation of Lamin A/C into progerin induces a senescent phenotype that inhibits the induction of reprogramming genes. Our results highlight the physical role of the nuclear scaffold in safeguarding cellular fate.

Magnesium ions mediate ligand binding and conformational transition of the SAM/SAH riboswitch

Hu, G. — 2023-03-12

The SAM/SAH riboswitch binds S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) with similar affinities. Mg2+ is generally known to stabilize RNA structures by neutralizing phosphates, but how it contributes to ligand binding and conformational transition is understudied. Here, extensive molecular dynamics simulations (totaling 120 s) identified over 10 inner-shell Mg2+ ions in the SAM/SAH riboswitch. Six of them line the two sides of a groove to widen it and thereby pre-organize the riboswitch for ligand entry. They also form outer-shell coordination with the ligands and stabilize an RNA-ligand hydrogen bond, which effectively diminish the selectivity between SAM and SAH. One Mg2+ ion unique to the apo form maintains the Shine-Dalgarno sequence in an autonomous mode and thereby facilitates its release for ribosome binding. Mg2+ thus plays vital roles in SAM/SAH riboswitch function.

Regulation of VEGFR2 and AKT signaling by Musashi-2 in lung cancer

Bychkov, I. — 2023-03-31

Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, expressed on both endothelial and tumor cells which is one of the key proteins contributing to cancer development and involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer which nominated VEGFR2 protein as strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human NSCLC cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human NSCLC samples. We conclude that MSI2/VEGFR2 axis contributes to NSCLC progression and is worth further investigations and therapeutic targeting.

Global Profiling of Differentiating Macrophages Identifies Novel Functional Long Non-coding RNAs Regulating Polarization and Innate Immune Responses

Valverde, A. — 2023-04-09

Macrophages (M{varphi}) are functionally dynamic immune cells that bridge innate and adaptive immune responses. However, the underlying epigenetic mechanisms that control the macrophage plasticity and innate immune functions are not well-elucidated. Here we performed transcriptome profiling of differentiating M1M{varphi} and M2M{varphi} and identified thousands of previously known and novel lncRNAs. We characterized three M{varphi}-enriched lncRNAs (LRRC75A-As1, GAPLINC and AL139099.5) with novel functions in M{varphi} differentiation, polarization and innate immunity. Knockdown of LRRC75A-As1, and GAPLINC downregulated M{varphi} differentiation markers CDw93 and CD68, and skewed macrophage polarization by decreasing M1 markers but had no significant impact on M2 markers. LRRC75A-As1, and GAPLINC RNAi in M{varphi} attenuated bacterial phagocytosis, antigen processing and inflammatory cytokine secretion supporting their functional role in potentiating innate immune functions. Mechanistically, lncRNA knockdown perturbed the expression of multiple cytoskeleton signaling thereby impairing M{varphi} migration suggesting their critical role in regulating macrophage polarity and motility. Together, our results show that M{varphi} acquire a unique repertoire of lncRNAs to shape differentiation, polarization and innate immune functions.

Matrisome AnalyzeR: A suite of tools to annotate and quantify ECM molecules in big datasets across organisms

Petrov, P. B. — 2023-04-20

The extracellular matrix (ECM) is a complex meshwork of proteins that forms the scaffold of all tissues in multicellular organisms. It plays critical roles in all aspects of life: from orchestrating cell migration during development, to supporting tissue repair. It also plays critical roles in the etiology or progression of diseases. To study this compartment, we defined the compendium of all genes encoding ECM and ECM-associated proteins for multiple organisms. We termed this compendium the "matrisome" and further classified matrisome components into different structural or functional categories. This nomenclature is now largely adopted by the research community to annotate -omics datasets and has contributed to advance both fundamental and translational ECM research. Here, we report the development of Matrisome AnalyzeR, a suite of tools including a web-based application (https://sites.google.com/uic.edu/matrisome/tools/matrisome-analyzer) and an R package (https://github.com/Matrisome/MatrisomeAnalyzeR). The web application can be used by anyone interested in annotating, classifying, and tabulating matrisome molecules in large datasets without requiring programming knowledge. The companion R package is available to more experienced users, interested in processing larger datasets or in additional data visualization options. SUMMARY STATEMENTMatrisome AnalyzeR is a suite of tools, including a web-based app and an R package, designed to facilitate the annotation and quantification of extracellular matrix components in big datasets.

Atomistic Modeling of Liquid-Liquid Phase Equilibrium Explains Dependence of Critical Temperature on γ-Crystallin Sequence

Qin, S. — 2023-04-28

Liquid-liquid phase separation of protein solutions has regained heightened attention for its biological importance and pathogenic relevance. Coarse-grained models are limited when explaining residue-level effects on phase equilibrium. Here we report phase diagrams for {gamma}-crystallins using atomistic modeling. The calculations were made possible by combining our FMAP method for computing chemical potentials and Brownian dynamics simulations for configurational sampling of dense protein solutions, yielding the binodal and critic temperature (Tc). We obtain a higher Tc for a known high-Tc {gamma}-crystallin, {gamma}F, than for a low-Tc paralog, {gamma}B. The difference in Tc is corroborated by a gap in second virial coefficient. Decomposition of inter-protein interactions reveals one amino-acid substitution between {gamma}B and {gamma}F, from Ser to Trp at position 130, as the major contributor to the difference in Tc. This type of analysis enables us to link phase equilibrium to amino-acid sequence and to design mutations for altering phase equilibrium.

Designer DNA NanoGripper

Zhou, L. — 2023-04-27

DNA has shown great biocompatibility, programmable mechanical properties, and structural addressability at the nanometer scale, making it a versatile material for building high precision nanorobotics for biomedical applications. Herein, we present design principle, synthesis, and characterization of a DNA nanorobotic hand, called the "NanoGripper", that contains a palm and four bendable fingers as inspired by human hands, bird claws, and bacteriophages evolved in nature. Each NanoGripper finger has three phalanges connected by two flexible and rotatable joints that are bendable in response to binding to other entities. Functions of the NanoGripper have been enabled and driven by the interactions between moieties attached to the fingers and their binding partners. We showcase that the NanoGripper can be engineered to interact with and capture various objects with different dimensions, including gold nanoparticles, gold NanoUrchins, and SARS-CoV-2 virions. When carrying multiple DNA aptamer nanoswitches programmed to generate fluorescent signal enhanced on a photonic crystal platform, the NanoGripper functions as a sensitive viral biosensor that detects intact SARS-CoV-2 virions in human saliva with a limit of detection of [~] 100 copies/mL, providing RT-PCR equivalent sensitivity. Additionally, we use confocal microscopy to visualize how the NanoGripper-aptamer complex can effectively block viral entry into the host cells, indicating the viral inhibition. In summary, we report the design, synthesis, and characterization of a complex nanomachine that can be readily tailored for specific applications. The study highlights a path toward novel, feasible, and efficient solutions for the diagnosis and therapy of other diseases such as HIV and influenza. One-sentence summaryDesign, synthesis, characterization, and functional showcase of a human-hand like designer DNA nanobot

Dimeric Transmembrane Structure of the SARS-CoV-2 E Protein

Zhang, R. — 2023-05-08

The SARS-CoV-2 E protein is a transmembrane (TM) protein with its N-terminus exposed on the external surface of the virus. Here, the TM structure of the E protein is characterized by oriented sample and magic angle spinning solid-state NMR in lipid bilayers and refined by molecular dynamics simulations. This protein has been found to be a pentamer, with a hydrophobic pore that appears to function as an ion channel. We identified only a symmetric helix-helix interface, leading to a dimeric structure that does not support channel activity. The two helices have a tilt angle of only 6{degrees}, resulting in an extended interface dominated by Leu and Val sidechains. While residues Val14-Thr35 are almost all buried in the hydrophobic region of the membrane, Asn15 lines a water-filled pocket that potentially serves as a drug-binding site. The E and other viral proteins may adopt different oligomeric states to help perform multiple functions.

Prostate-derived circulating microRNAs add prognostic value to prostate cancer risk calculators

Nonn, L. — 2023-05-12

Prostate cancer is the second leading cause of malignancy-related deaths among American men. Active surveillance is a safe option for many men with less aggressive disease, yet definitively determining low-risk cancer is challenging with biopsy alone. Herein, we sought to identify prostate-derived microRNAs in patient sera and serum extracellular vesicles, and determine if those microRNAs improve upon the current clinical risk calculators for prostate cancer prognosis before and after biopsy. Prostate-derived intracellular and extracellular vesicle-contained microRNAs were identified by small RNA sequencing of prostate cancer patient explants and primary cells. Abundant microRNAs were included in a custom microRNA PCR panel that was queried in whole serum and serum extracellular vesicles from a diverse cohort of men diagnosed with prostate cancer. The levels of these circulating microRNAs significantly differed between indolent and aggressive disease and improved the area under the curve for pretreatment nomograms of prostate cancer disease risk. The microRNAs within the extracellular vesicles had improved prognostic value compared to the microRNAs in the whole serum. In summary, quantifying microRNAs circulating in extracellular vesicles is a clinically feasible assay that may provide additional information for assessing prostate cancer risk stratification.

HOIL1 regulates group 3 innate lymphoid cell numbers in the colon and protects against systemic dissemination, colonic ulceration, and lethality from Citrobacter rodentium infection

Hartley, V. L. — 2023-05-24

HOIL1-deficient patients experience chronic intestinal inflammation and diarrhea as well as increased susceptibility to certain bacterial infections. HOIL1 is a component of the linear ubiquitin chain assembly complex (LUBAC) that regulates immune signaling pathways including NF-{kappa}B-activating pathways. We have shown previously that HOIL1 is essential for survival following Citrobacter rodentium gastrointestinal infection of mice, but the mechanism of protection by HOIL1 was not examined. C. rodentium is a murine model for human attaching and effacing (A/E) pathogens, enteropathogenic and enterohemorrhagic Escherichia coli, that cause diarrhea and food-borne illnesses, and lead to severe disease in children and immunocompromised individuals. In this study, we found that C. rodentium infection caused severe colitis and dissemination of C. rodentium to systemic organs in Hoil1-/-mice. HOIL1 was important in radiation-resistant cells and in the innate immune response to limit early replication of C. rodentium in the intestine, and to modulate induction of inflammatory cytokines. Using cell type-specific knock-out mice, we found that HOIL1 was dispensable in intestinal epithelial cells (IEC), but was required in CD11c- and lysozyme 2-expressing myeloid cells to prevent weight loss and systemic dissemination of C. rodentium. While HOIL1-deficiency did not affect populations of neutrophils or macrophages, dendritic cells and group 3 innate lymphoid cell (ILC3) numbers were reduced, resulting in a defect in IL-22 induction during C. rodentium infection. Understanding the role HOIL1 plays in limiting the pathogenesis of A/E lesion-forming bacteria will provide further insights into the innate immune response to gastrointestinal pathogens and inflammatory disorders.

Gel-assisted mass spectrometry imaging

Chan, Y. H. — 2023-06-03

Compatible with label-free detection and quantification, mass spectrometry imaging (MSI) is a powerful tool for spatial investigation of biomolecules in intact specimens. Yet, the spatial resolution of MSI is limited by the methods physical and instrumental constraints, which often preclude it from single-cell and subcellular applications. By taking advantage of the reversible interaction of analytes with superabsorbent hydrogels, we developed a sample preparation and imaging workflow named Gel-Assisted Mass Spectrometry Imaging (GAMSI) to overcome these limits. With GAMSI, the spatial resolution of lipid and protein MALDI-MSI can be enhanced severalfold without changing the existing mass spectrometry hardware and analysis pipeline. This approach will further enhance the accessibility to (sub)cellular-scale MALDI-MSI-based spatial omics.

A translational rodent model of individual differences in sensitivity to the aversive properties of ethanol

Przybysz, K. R. — 2023-06-09

BackgroundA strong relationship exists between individual sensitivity to the aversive properties of ethanol and risk for alcohol use disorder (AUD). Despite this, our understanding of the neurobiological mechanisms underlying subjective response to ethanol is relatively poor. A major contributor to this is the absence of preclinical models that enable exploration of this individual variability similar to studies performed in humans. MethodsAdult male and female Long-Evans rats were trained to associate a novel tastant (saccharin) with acute exposure to either saline or ethanol (1.5 g/kg or 2.0 g/kg i.p.) over three conditioning days using a standard conditioned taste aversion (CTA) procedure. Variability in sensitivity to ethanol-induced CTA was phenotypically characterized using a median split across the populations studied. ResultsWhen examining group averages, both male and female rats that had saccharin paired with either dose of ethanol exhibited reduced saccharin intake relative to saline controls of ethanol-induced CTA. Examination of individual data revealed a bimodal distribution of responses uncovering two distinct phenotypes present in both sexes. CTA-sensitive rats exhibited a rapid and progressive reduction in saccharin intake with each successive ethanol pairing. In contrast, saccharin intake was unchanged or maintained after an initial decrease from baseline levels in CTA-resistant rats. While CTA magnitude was similar between male and female CTA-sensitive rats, CTA-resistant females were more resistant to the development of ethanol-induced CTA than their male counterparts. Phenotypic differences were not driven by differences in baseline saccharin intake. CTA sensitivity correlated with behavioral signs of intoxication in only a subset of rats. ConclusionsThese data parallel work in humans by revealing individual differences in sensitivity to the aversive properties of ethanol that emerge immediately after initial exposure to ethanol in both sexes. This model can be leveraged in future studies to investigate the neurobiological mechanisms that confer risk for AUD.

Transcriptome data from silica-preserved leaf tissue reveals gene flow patterns in a Caribbean bromeliad

Ruiz-Vargas, N. — 2023-06-17

O_LITranscriptome sequencing is a cost-effective approach that allows researchers to study a broad range of questions. However, to preserve RNA for transcriptome sequencing, tissue is often kept under special conditions, such as immediate ultracold freezing. Here, we demonstrate that RNA can be obtained from six-month-old, field collected samples stored in silica gel at room temperature. Using these transcriptomes, we explore the evolutionary relationships of the genus Pitcairnia (Bromeliaceae) in the Dominican Republic and infer barriers to gene flow. C_LIO_LIWe extracted RNA from silica-dried leaf tissue from 19 Pitcairnia individuals collected across the Dominican Republic. We used a series of macro-and micro-evolutionary approaches to examine the relationships and patterns of gene flow among individuals. C_LIO_LIWe produced high-quality transcriptomes from silica-dried material and demonstrated that evolutionary relationships on the island match geography more closely than species delimitation methods. A population genetic examination indicates that a combination of ecological and geographic features are barriers to gene flow in Pitcairnia. C_LIO_LIHigh-quality transcriptomes can be obtained from silica-preserved tissue. The genetic diversity among Pitcairnia populations does not warrant classification as separate species, but the Dominican Republic contains several barriers to gene flow, notably the Cordillera Central mountain range. C_LI

A Common Pathway for Detergent-Assisted Oligomerization of Aβ42

Kunnath Muhammedkutty, F. N. — 2023-06-22

Amyloid beta (A{beta}) aggregation is a slow process without seeding or assisted nucleation. Sodium dodecyl sulfate (SDS) micelles stabilize A{beta}42 small oligomers (in the dimer-tetramer range); subsequent SDS removal leads to a 150-kD A{beta}42 oligomer. Dodecylphosphorylcholine (DPC) micelles also stabilize an A{beta}42 tetramer. Here we characterize the detergent-assisted oligomerization pathway by solid-state NMR spectroscopy and molecular dynamics simulations. SDS and DPC-induced oligomers have the same structure, implying a common oligomerization pathway. An antiparallel {beta}-sheet formed by the C-terminal region, the only stable structure in SDS and DPC micelles, is directly incorporated into the 150-kD oligomer. Three Gly residues (at positions 33, 37, and 38) create holes that are filled by the SDS and DPC hydrocarbon tails, thereby turning a potentially destabilizing feature into a stabilizing factor. These observations have implications for endogenous A{beta} aggregation at cellular interfaces.

Using energy to go downhill - a genoprotective role for ATPase activity in DNA topoisomerase II

Bandak, A. F. — 2023-06-27

SO_SCPLOWUMMARYC_SCPLOWType II topoisomerases effect topological changes in DNA by cutting a single duplex, passing a second duplex through the break, and resealing the broken strand in an ATP-coupled reaction. Curiously, most type II topoisomerases (topos II, IV, and VI) catalyze DNA transformations that are energetically favorable, such as the removal of superhelical strain; why ATP is required for such reactions is unknown. Here, using human topoisomerase II {beta} (hTOP2{beta}) as a model, we show that the ATPase domains of the enzyme are not required for DNA strand passage, but that their loss leads to increased DNA nicking and double strand break formation by the enzyme. The unstructured C-terminal domains (CTDs) of hTOP2{beta} strongly potentiate strand passage activity in the absence of the ATPase regions, as do cleavage-prone mutations that confer hypersensitivity to the chemotherapeutic agent etoposide. The presence of either the CTD or the mutations lead ATPase-less enzymes to promote even greater levels of DNA cleavaingevitro, as well as in vivo. By contrast, the aberrant cleavage phenotypes of these topo II variants is significantly repressed when the ATPase domains are restored. Our findings are consistent with the proposal that type II topoisomerases acquired an ATPase function to maintain high levels of catalytic activity while minimizing inappropriate DNA damage.

Elucidating the Impact of Bacterial Lipases, Human Serum Albumin, and FASII Inhibition on the Utilization of Exogenous Fatty Acids by Staphylococcus aureus

Pruitt, E. L. — 2023-06-29

Staphylococcus aureus only synthesizes straight-chain or branched-chain saturated fatty acids (SCFAs or BCFAs) via the type II fatty acid synthesis (FASII) pathway, but as a highly adaptive pathogen, S. aureus can also utilize host-derived exogenous fatty acids (eFAs), including SCFAs and unsaturated fatty acids (UFAs). S. aureus secretes three lipases, Geh, sal1, and SAUSA300_0641, which could perform the function of releasing fatty acids from host lipids. Once released, the FAs are phosphorylated by the fatty acid kinase, FakA, and incorporated into the bacterial lipids. In this study, we determined the substrate specificity of S. aureus secreted lipases, the effect of human serum albumin (HSA) on eFA incorporation, and the effect of FASII inhibitor, AFN-1252, on eFA incorporation using comprehensive lipidomics. When grown with major donors of fatty acids, cholesteryl esters (CEs) and triglycerides (TGs), Geh was found to be the primary lipase responsible for hydrolyzing CEs, but other lipases could compensate for the function of Geh in hydrolyzing TGs. Lipidomics showed that eFAs were incorporated into all major S. aureus lipid classes and that fatty acid-containing HSA can serve as a source of eFAs. Furthermore, S. aureus grown with UFAs displayed decreased membrane fluidity and increased production of reactive oxygen species (ROS). Exposure to AFN-1252 enhanced UFAs in the bacterial membrane, even without a source of eFAs, indicating a FASII pathway modification. Thus, the incorporation of eFAs alters the S. aureus lipidome, membrane fluidity, and ROS formation, which could affect host-pathogen interactions and susceptibility to membrane-targeting antimicrobials. IMPORTANCEIncorporation of host-derived exogenous fatty acids (eFAs), particularly unsaturated fatty acids (UFAs), by Staphylococcus aureus could affect the bacterial membrane fluidity and susceptibility to antimicrobials. In this work, we found that Geh is the primary lipase hydrolyzing cholesteryl esters and, to a less extent, triglycerides (TGs) and that human serum albumin (HSA) could serve as a buffer of eFAs, where low levels of HSA facilitate the utilization of eFAs, but high levels of HSA inhibit it. The fact that the FASII inhibitor, AFN-1252, leads to an increase in UFA content even in the absence of eFA suggests that membrane property modulation is part of its mechanism of action. Thus, Geh and/or the FASII system look to be promising targets to enhance S. aureus killing in a host environment by restricting eFA utilization or modulating membrane property, respectively.

Rgg2/Rgg3 quorum sensing is a determinant of Streptococcus pyogenes-host interactions in a murine intact skin infection model

Wilkening, R. V. — 2023-07-17

Streptococcus pyogenes is an obligate human pathobiont associated with many disease states. Here, we present a novel model of S. pyogenes infection using intact murine epithelium. From this model, we were able to perform RNA sequencing to evaluate the genetic changes undertaken by both the bacterium and host at 5- and 24-hours post infection. Analysis of these genomic data demonstrate that S. pyogenes undergoes significant genetic adaptation to successfully infect the murine epithelium, including changes to metabolism and activation of the Rgg2/Rgg3 quorum sensing (QS) system. Subsequent experiments demonstrate that an intact Rgg2/Rgg3 QS cascade is necessary to establish a stable superficial skin infection. Furthermore, activation of this pathway results in increased murine morbidity and increased bacterial burden on the skin. This phenotype is associated with gross changes to the murine skin, as well as histopathological evidence of inflammation. Taken together, these experiments offer a novel method to investigate S. pyogenes-epithelial interactions and demonstrate that a well-studied QS pathway is critical to a persistent infection. ImportanceStreptococcus pyogenes remains a pathogen of global importance, with significant total disease burden worldwide. Much of this burden is due to skin infection or sequalae thereof, yet little is currently known about the initial interactions between the organism and host skin. Here we present a new mouse model of skin infection. From this model, we were able to study gene expression by both the bacteria and the host during early infection time points. Both genetic and phenotypic data derived from these results demonstrate that a well-conserved S. pyogenes communication network, the Rgg2/Rgg3 quorum sensing pathway, contributes to establishing and maintaining a durable skin infection. We propose that by better understanding the genetic pathways needed to colonize and adapt to new niches, new approaches to preventing and treating S. pyogenes may be possible.

Microstructure-based Nuclear Lamina Constitutive Model

Mostafazadeh, N. — 2023-07-26

The nuclear lamina is widely recognized as the most crucial component in providing mechanical stability to the nucleus. However, it is still a significant challenge to model the mechanics of this multilayered protein network. We developed a constitutive model of the nuclear lamina network based on its microstructure, which accounts for the deformation phases at the dimer level, as well as the orientational arrangement and density of lamin filaments. Instead of relying on homology modeling in the previous studies, we conducted molecular simulations to predict the force-extension response of a highly accurate lamin dimer structure obtained through X-ray diffraction crystallography experimentation. Furthermore, we devised a semi-flexible worm-like chain extension-force model of lamin dimer as a substitute, incorporating phases of initial stretching, uncoiling of the dimer coiled-coil, and transition of secondary structures. Subsequently, we developed a 2D network continuum model for the nuclear lamina by using our extension-force lamin dimer model and derived stress resultants. By comparing with experimentally measured lamina modulus, we found that the lamina network has sharp initial strain-hardening behavior. This also enabled us to carry out finite element simulations of the entire nucleus with an accurate microstructure-based nuclear lamina model. Finally, we conducted simulations of transendothelial transmigration of a nucleus and investigated the impact of varying network density and uncoiling constants on the critical force required for successful transmigration. The model allows us to incorporate the microstructure characteristics of the nuclear lamina into the nucleus model, thereby gaining insights into how laminopathies and mutations affect nuclear mechanics.

Effect of the LSD1 Inhibitor RN-1 on gamma;-globin and Global Gene Expression during Erythroid Differentiation in Baboons (Papio anubis)

Lavelle, D. — 2023-07-28

Increased levels of Fetal Hemoglobin reduce the severity of symptoms of patients with sickle cell disease and decrease the risk of death. An affordable, small molecule drug that stimulates HbF expression in vivo would be ideally suited to treat the large numbers of SCD patients that exist worldwide. Our previous work showed that administration of the LSD1 (KDM1A) inhibitor RN-1 to normal baboons increased Fetal Hemoglobin (HbF) and was tolerated over a prolonged treatment period. HbF elevations were associated with changes in epigenetic modifications that included increased levels of H3K4 di-and tri-methyl lysine at the {gamma}-globin promoter. While dramatic effects of the loss of LSD1 on hematopoietic differentiation have been observed in murine LSD1 gene deletion and silencing models, the effect of pharmacological inhibition of LSD1 in vivo on hematopoietic differentiation is unknown. The goal of these experiments was to investigate the in vivo mechanism of action of the LSD1 inhibitor RN-1 by determining its effect on {gamma}-globin expression in highly purified subpopulations of bone marrow erythroid cells enriched for varying stages of erythroid differentiation isolated directly from baboons treated with RN-1 and also by investigating the effect of RN1 on the global transcriptome in a highly purified population of proerythroblasts. Our results show that RN-1 administered to baboons targets an early event during erythroid differentiation responsible for {gamma}-globin repression and increases the expression of a limited number of genes including genes involved in erythroid differentiation such as GATA2, GFi-1B, and LYN.

Ancient Mongolian aurochs genomes reveal sustained introgression and management in East Asia

Brunson, K. — 2023-08-10

Societies in East Asia have utilized domesticated cattle for over 5000 years, but the genetic history of cattle in East Asia remains understudied. Genome-wide analyses of 23 ancient Mongolian cattle reveal that East Asian aurochs and ancient East Asian taurine cattle are closely related, but neither are closely related to any modern East Asian breeds. We observe binary variation in aurochs diet throughout the early Neolithic, and genomic evidence shows millennia of sustained male-dominated introgression. We identify a unique connection between ancient Mongolian aurochs and the European Hereford breed. These results point to the likelihood of human management of aurochs in Northeast Asia prior to and during the initial adoption of taurine cattle pastoralism. One-Sentence SummaryAncient interbreeding of East Asian aurochs and cattle suggests management, but leaves no signature in modern eastern breeds.

Neuroendocrine control of synaptic transmission by PHAC-1 in C. elegans.

Stratigi, A. — 2023-08-20

A dynamic interplay between synaptic and neuromodulatory signalling guarantees flexible but robust neuronal circuits. Presynaptic modulation plays a crucial role in controlling the excitatory-inhibitory balance within networks. Here, we designed a genetic screen to identify genes involved in the neuromodulation of the C. elegans neuromuscular junctions (NMJ) and identified the orthologs of the Protein Phosphatase 1 regulatory subunit PHACTR1 (phac-1) and the presynaptic phosphoproteins Synapsin (snn-1). Five de novo variants of human PHACTR1 are associated with severe early-onset epilepsies (DEE70). To understand the impact of these variants, we introduced the DEE70 mutations into phac-1. These mutations resulted in the formation of a constitutively active PP1-PHAC-1 holoenzyme that disrupts cholinergic signalling at the NMJ. By using quantitative fluorescence imaging, electron microscopy and electrophysiology, we found that the constitutive holoenzyme alters the synaptic vesicle cycle, reduces the synaptic vesicle reserve pool, and increases neuropeptide release by dense-core vesicles. Notably, while SNN-1 phosphoregulation contributes to NMJ signalling, genetic interactions suggest that SNN-1 is not the main effector of PP1-PHAC-1 holoenzyme signalling. Collectively, our results confirm the pathogenicity of DEE70 variants, clarify their dominant-positive effects, and provide evidence of a presynaptic mode of action for DEE70.

ATP Mediates Phase Separation of Disordered Basic Proteins by Bridging Intermolecular Interaction Networks

Kota, D. — 2023-08-20

ATP is an abundant molecule with crucial cellular roles as the energy currency and a building block of nucleic acids and for protein phosphorylation. Here we show that ATP mediates the phase separation of basic intrinsically disordered proteins (bIDPs). In the resulting condensates, ATP is highly concentrated (apparent partition coefficients at 200-5000) and serves as bridges between bIDP chains. These liquid-like droplets have some of the lowest interfacial tension ([~]25 pN/m) but high zero-shear viscosities (1-15 Pa s) due to the bridged protein networks, and yet their fusion has some of the highest speeds ([~]1 m/ms). The rapid fusion manifests extreme shear thinning, where the apparent viscosity is lower than zero-shear viscosity by over 100-fold, made possible by fast reformation of the ATP bridges. At still higher concentrations, ATP does not dissolve bIDP droplets but results in aggregates and fibrils.

An autochthonous model of lung cancer in the Naked Mole-Rat (Heterocephalus glaber)

Kissil, J. — 2023-08-28

Studies on cancer resistance in the naked mole-rat (NMR) have generally failed to interrogate possible resistance mechanisms in a physiological context. Here, we provide evidence that the NMR presents as a novel model of tumor initiation. We developed an endogenous lung cancer model in NMRs, driven by an oncogenic Eml4-Alk fusion protein introduced through CRISPR- mediated genome editing. While this is sufficient to drive tumorigenesis in mice, the development of progressive disease in NMRs required the additional loss of key tumor suppressors. Our results show that tumor initiation in NMRs more closely recapitulates that of human tumors. This suggests that the proposed "resistance" of NMRs to cancer development may stem from tumor initiation events that are likely to be comparable to the mechanisms in human cells. One-Sentence SummaryTumor development in the cancer-resistant naked mole-rat more accurately represents the tumor initiation process in humans.

Murine leukemia virus can infect non-dividing primary cells

Salas-Briceno, K. — 2023-08-30

Retroviral reverse transcription starts within the capsid and uncoating and reverse transcription are mutually dependent. There is still debate regarding the timing and cellular location of HIVs uncoating and reverse transcription and whether it occurs solely in the cytoplasm, nucleus or both. HIV can infect non-dividing cells because there is active transport of the preintegration complex (PIC) across the nuclear membrane, but Murine Leukemia Virus (MLV) is thought to depend on cell division for replication and whether MLV uncoating and reverse transcription is solely cytoplasmic has not been studied. Here, we used NIH3T3 and primary mouse dendritic cells to determine where the different stages of reverse transcription occur and whether cell division is needed for nuclear entry. Our data strongly suggest that in both NIH3T3 cells and dendritic cells (DCs), the initial step of reverse transcription occurs in the cytoplasm. However, we detected MLV RNA/DNA hybrid intermediates in the nucleus of dividing NIH3T3 cells and non-dividing DCs, suggesting that reverse transcription can continue after nuclear entry. We also found that the MLV PIC requires cell division to enter the nucleus of NIH3T3 cells. In contrast, we show that MLV can infect non-dividing primary DCs, although integration of MLV DNA in DCs still required the viral p12 protein. Knockdown of several nuclear pore proteins dramatically reduced the appearance of integrated MLV DNA in DCs but not NIH3T3 cells. Additionally, MLV capsid associates with the nuclear pore proteins NUP358 and NUP62 during infection. These findings suggest that simple retroviruses, like HIV, gain nuclear entry by traversing the nuclear pore complex in non-mitotic cells. Author SummaryIt is widely believed that gammaretroviruses like MLV require cell division to achieve nuclear entry and complete their replication. We show here that while this is true for rapidly dividing tissue culture cells, in quiescent cells like dendritic cells, the natural targets of MLV infection, the virus establishes infection without cell division. These studies show that the requirements for retrovirus infection depend on the cell type.

Claudin-2 limits pancreatitis development through regulating tight junction-controlled pancreatic ductal transport

Kesaraju, S. — 2023-09-05

Pancreatitis is an inflammatory disease of the pancreas that can arise due to various factors, including environmental risks such as diet, alcohol, and smoking, as well as genetic predispositions. In some cases, pancreatitis may progress and become chronic, leading to irreversible damage and impaired pancreatic function. Genome-wide association studies (GWAS) have identified polymorphisms at the X-linked CLDN2 locus as risk factors for both sporadic and alcohol-related chronic pancreatitis. CLDN2 encodes claudin-2 (CLDN2), a paracellular cation-selective channel localized at tight junctions and expressed in the pancreas and other secretory organs. However, whether and how CLDN2 may modify pancreatitis susceptibility remains poorly understood. We aimed to clarify the potential role of CLDN2 in the onset and progression of pancreatitis. We employed multiple methodologies to examine the role of CLDN2 in human pancreatic tissue, caerulein-induced experimental pancreatitis mouse model, and pancreatic ductal epithelial organoids. In both human chronic pancreatitis tissues and caerulein-induced experimental pancreatitis, CLDN2 protein was significantly upregulated in pancreatic ductal epithelial cells. Our studies using pancreatic ductal epithelial organoids and mice demonstrated the inflammatory cytokine IFN{gamma} upregulates claudin-2 expression at both RNA and protein levels. Following caerulein treatment, Ifng KO mice had diminished upregulation of CLDN2 relative to WT mice, indicating that caerulein-induced claudin-2 expression is partially driven by IFN{gamma}. Functionally, Cldn2 knockout mice developed more severe caerulein-induced experimental pancreatitis, indicating CLDN2 plays a protective role in pancreatitis development. Pancreatic ductal epithelial organoid-based studies demonstrated that CLDN2 is critical for sodium-dependent water transport and necessary for cAMP-driven, CFTR-dependent fluid secretion. These findings suggest that functional crosstalk between CLDN2 and CFTR is essential for fluid transport in pancreatic ductal epithelium, which may protect against pancreatitis by adjusting pancreatic ductal secretion to prevent worsening autodigestion and inflammation. In conclusion, our studies suggest CLDN2 upregulation during pancreatitis may play a protective role in limiting disease development, and decreased CLDN2 function may increase pancreatitis severity. These results point to the possibility of modulating pancreatic ductal CLDN2 function as an approach for therapeutic intervention of pancreatitis.

Photoemission electron microscopy for connectomics

Boergens, K. M. — 2023-09-06

1Detailing the physical basis of neural circuits with large-volume serial electron microscopy (EM), connectomics, has emerged as an invaluable tool in the neuroscience armamentarium. However, imaging synaptic resolution connectomes is currently limited to either transmission electron microscopy (TEM) or scanning electron microscopy (SEM). Here, we describe a third way, using photoemission electron microscopy (PEEM) which illuminates ultra-thin brain slices collected on solid substrates with UV light and images the photoelectron emission pattern with a wide-field electron microscope. PEEM works with existing sample preparations for EM and routinely provides sufficient resolution and contrast to reveal myelinated axons, somata, dendrites, and sub-cellular organelles. Under optimized conditions, PEEM provides synaptic resolution; and simulation and experiments show that PEEM can be transformatively fast, at Gigahertz pixel rates. We conclude that PEEM imaging leverages attractive aspects of SEM and TEM, namely reliable sample collection on robust substrates combined with fast wide-field imaging, and could enable faster data acquisition for next-generation circuit mapping.

Genetic synergy in Acinetobacter baumannii undecaprenyl biosynthesis and maintenance of lipid asymmetry impacts outer membrane and antibiotic resistance

Noel, H. R. — 2023-09-22

Acinetobacter baumannii is a Gram-negative healthcare-associated pathogen that poses a major health concern due to increasing multidrug resistance. The Gram-negative cell envelope is a key barrier to antimicrobial entry and includes an inner and outer membrane. The outer membrane has an asymmetric composition that is important for structural integrity and barrier to the environment. Therefore, Gram-negative bacteria have mechanisms to uphold this asymmetry such as the maintenance of lipid asymmetry system (Mla), which removes glycerophospholipids from the outer leaflet of the outer membrane and transports them to the inner membrane. Loss of this system in A. baumannii results in attenuated virulence and increased susceptibility to membrane stressors and some antibiotics. We recently reported two strain variants of the A. baumannii type strain ATCC 17978, 17978VU and 17978UN. We show here that {Delta}mlaF mutants in the two strains display different phenotypes for membrane stress resistance, antibiotic resistance, and pathogenicity in a murine pneumonia model. We used comparative genetics to identify interactions between ATCC 17978 strain alleles and mlaF to uncover the cause behind the phenotypic differences. Although allele differences in obgE were previously reported to synergize with {Delta}mlaF to affect growth and stringent response, we show that obgE alleles do not affect membrane stress resistance. Instead, a single nucleotide polymorphism (SNP) in the essential gene encoding undecaprenyl pyrophosphate (Und-PP) synthase, uppS, synergizes with {Delta}mlaF to increase susceptibility to membrane stress and antibiotics, and reduce persistence in a mouse lung infection. Und-P is a lipid glycan carrier known to be required for biosynthesis of A. baumannii capsule, cell wall, and glycoproteins. Our data suggest that in the absence of the Mla system, the cellular level of Und-P is critical for envelope integrity, antibiotic resistance, and lipooligosaccharide abundance. These findings uncover synergy between Und-P and the Mla system in maintaining the A. baumannii outer membrane and stress resistance.

Caveolin-1 Autonomously Regulates Hippocampal Neurogenesis Via Mitochondrial Dynamics

Stephen, T. K. L. — 2023-09-24

Hippocampal neurogenesis plays instrumental roles in learning and memory. However, the mechanisms underlying neurogenesis are not fully understood. Here we show that the expression of Caveolin-1 (Cav-1), the principal component of caveolae, peaks in neural progenitor cells (NPCs) during neurogenesis. Using NestinCreERT2;Cav-1fl/fl male mice, and CRISPR-sham (Cav-1 Ctrl) and CRISPR/Cas9-edited (Cav-1 KO) human induced-pluripotent stem cells, we observed that Cav-1 deletion led to reduced stem cell proliferation and enhanced differentiation into neurons. This was manifested by increased neuronal dendritic tree surface area and enhanced mouse performance in contextual discrimination. Proteomic analysis revealed that Cav-1 plays a role in mitochondrial pathways in NPCs. Cav-1 localized to the mitochondria in NPCs and co-immunoprecipitated with mitofusion 2. Mitochondrial morphology was elongated in Cav-1 KO NPCs and the expression of mitofusion 2 was increased in mitochondrial fractions. Restoration of Cav-1 levels rescued elongated mitochondrial morphology and altered neuronal differentiation in Cav-1 KO NPCs. Together, this study identifies Cav-1 as a novel regulator of neurogenesis and -dependent learning and memory. Significance StatementThe hippocampal dentate gyrus (DG) orchestrates adult hippocampal neurogenesis (AHN). The precise mechanisms governing AHN remain elusive. Caveolin-1 regulates neurogenesis through mitochondrial fission-fusion process, suggesting Caveolin-1 as a novel regulator of AHN and underscoring the impact of AHN on cognition.

A Novel Subtype of Myeloproliferative Neoplasms Driven by a MYC-Alarmin Axis

Vincelette, N. D. — 2023-09-26

Despite advances in understanding the genetic abnormalities in myeloproliferative neoplasms (MPNs) and the development of JAK2 inhibitors, there is an urgent need to devise new treatment strategies, particularly for triple negative myelofibrosis (MF) patients whose MPNs lack mutations in the JAK2 kinase pathway and have very poor clinical outcomes. Here we report that MYC copy number gain and increased MYC expression frequently occur in triple negative MF, and that MYC-directed activation of S100A9, an alarmin protein that plays pivotal roles in inflammation and innate immunity, is necessary and sufficient to drive development and progression of MF. Notably, the MYC-S100A9 circuit provokes a complex network of inflammatory signaling that involves various hematopoietic cell types in the bone marrow microenvironment. Accordingly, genetic ablation of S100A9 or treatment with small molecules targeting the MYC-S100A9 pathway effectively ameliorates MF phenotypes, highlighting the MYC-alarmin axis as a novel therapeutic vulnerability for this subgroup of MPNs. SIGNIFICANCEThis study establishes that MYC expression is increased in triple negative MPNs via trisomy 8, that a MYC-S100A9 circuit manifest in these cases is sufficient to provoke myelofibrosis and inflammation in diverse hematopoietic cell types in the BM niche, and that the MYC-S100A9 circuit is targetable in triple negative MPN.

Identifying representational structure in CA1 to benchmark theoretical models of cognitive mapping

Lee, J. Q. — 2023-10-08

Decades of theoretical and empirical work have suggested the hippocampus instantiates some form of a cognitive map. Yet, tests of competing theories have been limited in scope and largely qualitative in nature. Here, we develop a novel framework to benchmark model predictions against observed neuronal population dynamics as animals navigate a series of geometrically distinct environments. In this task space, we show a representational structure in the dynamics of hippocampal remapping that generalizes across brains, discriminates between competing theoretical models, and effectively constrains biologically viable model parameters. With this approach, we find that accurate models capture the correspondence in spatial coding of a changing environment. The present dataset and framework thus serve to empirically evaluate and advance theories of cognitive mapping in the brain. HIGHLIGHTSO_LIWe identify representational structure in CA1 remapping that is reliable across brains. C_LIO_LIWe directly compare models of cognitive mapping to this representation in CA1. C_LIO_LIModels based on local boundary distance and direction predict CA1 representation. C_LIO_LIThis approach reveals a biologically viable parameter space for model predictions. C_LIO_LIAccurate models capture the correspondence of spatial codes across environments. C_LI

Distinct information conveyed to the olfactory bulb by feedforward input from the nose and feedback from the cortex

Zak, J. D. — 2023-10-10

Sensory systems are organized hierarchically, but feedback projections frequently disrupt this order. In the olfactory bulb (OB), cortical feedback projections numerically match sensory inputs. To unravel information carried by these two streams, we imaged the activity of olfactory sensory neurons (OSNs) and cortical axons in the mouse OB using calcium indicators, multiphoton microscopy, and diverse olfactory stimuli. Odorant mixtures of increasing complexity evoked progressively denser OSN activity, yet cortical feedback activity was of similar sparsity for all stimuli. Representations of complex mixtures were similar in OSNs but were decorrelated in cortical axons. While OSN responses to increasing odorant concentrations exhibited a sigmoidal relationship, cortical axonal responses were complex and non-monotonic, which could be explained by a model with activity-dependent feedback inhibition in the cortex. Our study indicates that early-stage olfactory circuits have access to both local feedforward signals and global, efficiently formatted information about odor scenes through cortical feedback.

Oral copper-methionine decreases matrix metalloproteinase-2 activity in the liver and brain of broiler chickens subjected to cold stress for ascites incidence

Bagheri Varzaneh, M. — 2023-10-15

Copper plays an antioxidant role in biological reactions. This study examined the impact of copper-methionine supplementation on the matrix metalloproteinase-2 (MMP-2) activity and gene expression in the liver and brain of broiler chickens subjected to cold temperature. A total of 480 broiler chickens were assigned to 6 groups and reared under either low (15-19 {o}C) or normal temperature (25-28{o}C) and fed a basal diet enriched with different concentrations of copper-methionine (Cu-Met) supplementation (0, 100 or 200 mg.kg-1). Ascites was exclusively observed in broiler chickens kept in low temperature and fed with basal diet without Cu-Met during the seventh week, identified by the presence of abdominal fluid accumulation. Broilers livers and brains were separated for MMP-2 and tissue inhibitor of metalloproteinase-2 (TIMP-2) analysis. Results of gelatin zymography on these samples demonstrated that incidence of ascites was associated with increased MMP-2 levels in liver and brain. MMP-2 activity assay confirmed the results obtained by zymography. RT-qPCR experiments revealed an upregulation in the mRNA expression of MMP-2. In contrast, the treatments did not induce significant alterations in TIMP-2 levels. Results suggest that oral copper-methionine can decrease the ascites occurrence and might be useful for prevention of ascites in broiler chickens.

A statistical framework for powerful multi-trait rare variant analysis in large-scale whole-genome sequencing studies

Li, X. — 2023-11-02

Large-scale whole-genome sequencing (WGS) studies have improved our understanding of the contributions of coding and noncoding rare variants to complex human traits. Leveraging association effect sizes across multiple traits in WGS rare variant association analysis can improve statistical power over single-trait analysis, and also detect pleiotropic genes and regions. Existing multi-trait methods have limited ability to perform rare variant analysis of large-scale WGS data. We propose MultiSTAAR, a statistical framework and computationally-scalable analytical pipeline for functionally-informed multi-trait rare variant analysis in large-scale WGS studies. MultiSTAAR accounts for relatedness, population structure and correlation among phenotypes by jointly analyzing multiple traits, and further empowers rare variant association analysis by incorporating multiple functional annotations. We applied MultiSTAAR to jointly analyze three lipid traits (low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides) in 61,861 multi-ethnic samples from the Trans-Omics for Precision Medicine (TOPMed) Program. We discovered new associations with lipid traits missed by single-trait analysis, including rare variants within an enhancer of NIPSNAP3A and an intergenic region on chromosome 1.

Microscale visualization of cellular features in adult macaque visual cortex

Balaram, P. — 2023-11-05

Expansion microscopy and light sheet imaging enable fine-scale resolution of intracellular features that comprise neural circuits. Most current techniques visualize sparsely distributed features across whole brains or densely distributed features within individual brain regions. Here, we visualize dense distributions of immunolabeled proteins across early visual cortical areas in adult macaque monkeys. This process may be combined with multiphoton or magnetic resonance imaging to produce multimodal atlases in large, gyrencephalic brains.

Whole genome association testing in 333,100 individuals across three biobanks identifies rare non-coding single variant and genomic aggregate associations with height

Hawkes, G. — 2023-11-20

The role of rare non-coding variation in complex human phenotypes is still largely unknown. To elucidate the impact of rare variants in regulatory elements, we performed a whole-genome sequencing association analysis for height using 333,100 individuals from three datasets: UK Biobank (N=200,003), TOPMed (N=87,652) and All of Us (N=45,445). We performed rare (<0.1% minor-allele-frequency) single-variant and aggregate testing of non-coding variants in regulatory regions based on proximal, intergenic and deep-intronic annotation. We observed 29 independent variants associated with height at P < 6 x 10-10 after conditioning on previously reported variants, with effect sizes ranging from -7cm to +4.7cm. We also identified and replicated non-coding aggregate-based associations proximal to HMGA1 containing variants associated with a 5cm taller height and of highly-conserved variants in MIR497HG on chromosome 17. We have developed a novel approach for identifying non-coding rare variants in regulatory regions with large effects from whole-genome sequencing data associated with complex traits.

Increased multiplexity in optical tissue clearing-based 3D immunofluorescence microscopy of the tumor microenvironment by LED photobleaching

zheng, j. — 2023-12-01

Optical tissue clearing and three-dimensional (3D) immunofluorescence (IF) microscopy have been transforming imaging of the complex tumor microenvironment (TME). However, current 3D IF microscopy has restricted multiplexity; only three or four cellular and non-cellular TME components can be localized in a cleared tumor tissue. Here we report a LED photobleaching method and its application for 3D multiplexed optical mapping of the TME. We built a high-power LED light irradiation device and temperature-controlled chamber for completely bleaching fluorescent signals throughout optically cleared tumor tissues without compromise of tissue and protein antigen integrity. With newly developed tissue mounting and selected region-tracking methods, we established a cyclic workflow involving IF staining, tissue clearing, 3D confocal microscopy, and LED photobleaching. By registering microscope channel images generated through three work cycles, we produced 8-plex image data from individual 400 m-thick tumor macrosections that visualize various vascular, immune, and cancer cells in the same TME at tissue-wide and cellular levels in 3D. Our method was also validated for quantitative 3D spatial analysis of cellular remodeling in the TME after immunotherapy. These results demonstrate that our LED photobleaching system and its workflow offer a novel approach to increase the multiplexing power of 3D IF microscopy for studying tumor heterogeneity and response to therapy.

Multiple modes of DNA compaction by protamine

Ahlawat, V. — 2023-12-08

In sperm cells, protamine replaces histones to compact DNA 10-20 times more than in somatic cells. To characterize the extreme compaction, we employed confocal microscopy and optical tweezers to determine the conformations and stability of protamine-bound {lambda}-DNA. Confocal images show increasing compaction of {lambda}-DNA at increasing protamine concentration. In the presence of protamine, single {lambda}-DNA molecules form bends and loops that unravel at 10-40 pN forces as well as coils that shorten the contour length by up to 40% and withstand forces strong enough ([~]55 pN) for strand separation. Strand separation nucleates coils, indicating protamine insertion into DNA bases. Protamine may participate in both local and higher-order chromatin organization, leading to extreme compaction and global transcription silencing. One-Sentence SummaryProtamine compacts sperm DNA in multiple modes, producing bends and loops but also coils that may block transcription.

IIHP: Intelligent Incident Hypertension Prediction in Obstructive Sleep Apnea

Halimi Milani, O. — 2023-12-15

The Obstructive sleep apnea (OSA) increases the risk of hypertension, mainly attributed to intermittent hypoxia and sleep fragmentation. Given the multifaceted pathogenesis of hypertension, accurately predicting incident hypertension in individuals with OSA has posed a considerable challenge. In this study, we leveraged Machine Learning (ML) techniques to develop a predictive model for incident hypertension up to five years after OSA diagnosis by polysomnography. We used data from the Sleep Heart Health Study (SHHS), which included 4,797 participants diagnosed with OSA. After excluding those with pre-existing hypertension and Apnea Hypopnea Index (AHI) values below 21 per hour, we had 671 participants with five-year follow-up data. We adopted two distinct methodologies. We first implemented adaptive convolution layers to extract features from the signals and combined them into a 2D array. The 2D array was further processed by a 2D pre-trained neural network to take advantage of transfer learning. Subsequently, we delved into feature extraction from full-length signals across various temporal frames, resulting in a 2D feature array. We studied the use of various 2D networks such as MobileNet, EfficientNet, and a family of RESNETs. The best algorithm achieved an average area under the curve of 72%. These results suggest a promising approach for predicting the risk of incident hypertension in individuals with OSA, providing tools for practice and public health initiatives.

Parental infections disrupt clustered genes encoding related functions required for nervous system development in newborns

Friedenson, B. — 2018-10-22

The purpose of this study was to understand the role of infection in the origin of chromosomal anomalies linked to neurodevelopmental disorders. In children with disorders in the development of their nervous systems, chromosome anomalies known to cause these disorders were compared to viruses and bacteria including known teratogens. Results support the explanation that parental infections disrupt elaborate multi-system gene coordination needed for neurodevelopment. Genes essential for neurons, lymphatic drainage, immunity, circulation, angiogenesis, cell barriers, structure, and chromatin activity were all found close together in polyfunctional clusters that were deleted in neurodevelopmental disorders. These deletions account for immune, circulatory, and structural deficits that accompany neurologic deficits. In deleted gene clusters, specific and repetitive human DNA matched infections and passed rigorous artifact tests. In some patients, epigenetic driver mutations were found and may be functionally equivalent to deleting a cluster or changing topologic chromatin interactions because they change access to large chromosome segments. In three families, deleted DNA sequences were associated with intellectual deficits and were not included in any database of genomic variants. These sequences were thousands of bp and unequivocally matched foreign DNAs. Analogous homologies were also found in chromosome anomalies of a recurrent neurodevelopmental disorder. Viral and bacterial DNAs that match repetitive or specific human DNA segments are thus proposed to interfere with highly active break repair during meiosis; sometimes delete polyfunctional clusters, and disable epigenetic drivers. Mis-repaired gametes produce zygotes containing rare chromosome anomalies which cause neurologic disorders and accompanying non-neurologic signs. Neurodevelopmental disorders may be examples of assault on the human genome by foreign DNA with some infections more likely tolerated because they resemble human DNA segments. Further tests of this model await new technology.nnGraphic AbstractnnO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/448845_ufig1.gif" ALT="Figure 1">nView larger version (55K):norg.highwire.dtl.DTLVardef@1c1bf87org.highwire.dtl.DTLVardef@1056721org.highwire.dtl.DTLVardef@1b5ae34org.highwire.dtl.DTLVardef@c007fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

A Genome Model Linking Birth Defects to Infections

Friedenson, B. A. — 2019-06-19

The purpose of this study was to test the hypothesis that infections are linked to chromosomal anomalies that cause neurodevelopmental disorders. In children with disorders in the development of their nervous systems, chromosome anomalies known to cause these disorders were compared to microbial DNA, including known teratogens. Genes essential for neurons, lymphatic drainage, immunity, circulation, angiogenesis, cell barriers, structure, epigenetic and chromatin modifications were all found close together in polyfunctional clusters that were deleted or rearranged in neurodevelopmental disorders. In some patients, epigenetic driver mutations also changed access to large chromosome segments. These changes account for immune, circulatory, and structural deficits that accompany neurologic deficits. Specific and repetitive human DNA encompassing large deletions matched infections and passed rigorous artifact tests. Deletions of up to millions of bases accompanied infection-matching sequences and caused massive changes in the homologies to foreign DNAs. In data from three independent studies of private, familial and recurrent chromosomal rearrangements, massive changes in homologous microbiomes were found and may drive rearrangements and encourage pathogens. At least one chromosomal anomaly was found to consist of human DNA fragments with a gap that corresponded to a piece of integrated foreign DNA. Microbial DNAs that match repetitive or specific human DNA segments are thus proposed to interfere with the epigenome and highly active recombination during meiosis, driven by massive changes in the homologous microbiome. Abnormal recombination in gametes produces zygotes containing rare chromosome anomalies which cause neurologic disorders and non-neurologic signs. Neurodevelopmental disorders may be examples of assault on the human genome by foreign DNA at a critical stage. Some infections may be more likely tolerated because they resemble human DNA segments. Further tests of this model await new technology.nnnnO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/674093v1_ufig1.gif" ALT="Figure 1">nView larger version (47K):norg.highwire.dtl.DTLVardef@a929b4org.highwire.dtl.DTLVardef@1f10ba3org.highwire.dtl.DTLVardef@331a1aorg.highwire.dtl.DTLVardef@f6af6c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ancient human genomes suggest three ancestral populations for present-day Europeans

Iosif Lazaridis — 2013-12-23

We sequenced genomes from a [~]7,000 year old early farmer from Stuttgart in Germany, an [~]8,000 year old hunter-gatherer from Luxembourg, and seven [~]8,000 year old hunter-gatherers from southern Sweden. We analyzed these data together with other ancient genomes and 2,345 contemporary humans to show that the great majority of present-day Europeans derive from at least three highly differentiated populations: West European Hunter-Gatherers (WHG), who contributed ancestry to all Europeans but not to Near Easterners; Ancient North Eurasians (ANE), who were most closely related to Upper Paleolithic Siberians and contributed to both Europeans and Near Easterners; and Early European Farmers (EEF), who were mainly of Near Eastern origin but also harbored WHG-related ancestry. We model these populations deep relationships and show that EEF had [~]44% ancestry from a "Basal Eurasian" lineage that split prior to the diversification of all other non-African lineages.

Preparation of Synaptosomes from Postmortem Human Prefrontal Cortex

Neil R Smalheiser — 2014-06-24

Synaptosomes are a popular type of isolated synaptic fraction intensively used in neuroscience and cell biology. They are prepared by layering on density gradients and thought to consist largely of axonal endings with attached postsynaptic structures (Morgan, 1976), in contrast to synaptoneurosomes (Hollingsworth et al, 1985) which are prepared by filtration and are thought to consist largely of pinched-off dendritic spines with attached presynaptic structures. Although most studies of synaptosomes have utilized rodent or primate tissue, a score of studies have employed human samples derived from surgical specimens or postmortem brain. We recently described the isolation of synaptosomes from human postmortem prefrontal cortex to study the expression of synaptic microRNAs and other small RNAs in depression, schizophrenia and bipolar disorder (Smalheiser et al, 2014). This protocol alluded to methods and modifications that are scattered among several publications, and did not explain the reasons for the procedures chosen. Because our protocol differs from other published human synaptosome protocols in a variety of respects, we present here a detailed description of synaptosome preparation that should facilitate the use of this standardized synaptic fraction by other workers.

Deep, Staged Transcriptomic Resources for the Novel Coleopteran Models Atrachya menetriesi and Callosobruchus maculatus

Matthew Alan Benton — 2016-01-06

Despite recent efforts to sample broadly across metazoan and insect diversity, current sequence resources in the Coleoptera do not adequately describe the diversity of the clade. Here we present deep, staged transcriptomic data for two coleopteran species, Atrachya menetriesi (Faldermann 1835) and Callosobruchus maculatus (Fabricius 1775). Our sampling covered key stages in ovary and early embryonic development in each species. We utilized this data to build combined assemblies for each species which were then analysed in detail. The combined A. menetriesi assembly consists of 228,096 contigs with an N50 of 1,598 bp, while the combined C. maculatus assembly consists of 128,837 contigs with an N50 of 2,263 bp. For these assemblies, 34.6% and 32.4% of contigs were identified using Blast2GO, and 97% and 98.3% of the BUSCO set of metazoan orthologs were present, respectively. We also carried out manual annotation of developmental signalling pathways and found that nearly all expected genes were present in each transcriptome. Our analyses show that both transcriptomes are of high quality. Lastly, we performed read mapping utilising our timed, stage specific RNA samples to identify differentially expressed contigs. The resources presented here will provide a firm basis for a variety of experimentation, both in developmental biology and in comparative genomic studies.

Two Similarity Metrics for Medical Subject Headings (MeSH): An Aid to Biomedical Text Mining and Author Name Disambiguation

Neil R Smalheiser — 2016-02-06

In the present paper, we have created and characterized several similarity metrics for relating any two Medical Subject Headings (MeSH terms) to each other. The article-based metric measures the tendency of two MeSH terms to appear in the MEDLINE record of the same article. The author-based metric measures the tendency of two MeSH terms to appear in the body of articles written by the same individual (using the 2009 Author-ity author name disambiguation dataset as a gold standard). The two metrics are only modestly correlated with each other (r = 0.50), indicating that they capture different aspects of term usage. The article-based metric provides a measure of semantic relatedness, and MeSH term pairs that co-occur more often than expected by chance may reflect relations between the two terms. In contrast, the author metric is indicative of how individuals practice science, and may have value for author name disambiguation and studies of scientific discovery. We have calculated article metrics for all MeSH terms appearing in at least 25 articles in MEDLINE (as of 2014) and author metrics for MeSH terms published as of 2009. The dataset is freely available for download and can be queried at http://arrowsmith.psych.uic.edu/arrowsmith_uic/mesh_pair_metrics.html.

Genome-wide association study of 40,000 individuals identifies two novel loci associated with bipolar disorder

Liping Hou — 2016-03-22

Bipolar disorder (BD) is a genetically complex mental illness characterized by severe oscillations of mood and behavior. Genome-wide association studies (GWAS) have identified several risk loci that together account for a small portion of the heritability. To identify additional risk loci, we performed a two-stage meta-analysis of >9 million genetic variants in 9,784 bipolar disorder patients and 30,471 controls, the largest GWAS of BD to date. In this study, to increase power we used ~2,000 lithium-treated cases with a long-term diagnosis of BD from the Consortium on Lithium Genetics, excess controls, and analytic methods optimized for markers on the X-chromosome. In addition to four known loci, results revealed genome-wide significant associations at two novel loci: an intergenic region on 9p21.3 (rs12553324, p = 5.87x10-9; odds ratio = 1.12) and markers within ERBB2 (rs2517959, p = 4.53x10-9; odds ratio = 1.13). No significant X-chromosome associations were detected and X-linked markers explained very little BD heritability. The results add to a growing list of common autosomal variants involved in BD and illustrate the power of comparing well-characterized cases to an excess of controls in GWAS.

WEVOTE: Weighted Voting Taxonomic Identification Method of Microbial Sequences

Ahmed A. Metwally — 2016-05-19

Metagenome shotgun sequencing presents opportunities to identify organisms that may prevent or promote disease. The analysis of sample diversity is achieved by taxonomic identification of metagenomic reads followed by generating an abundance profile. Numerous tools have been developed based on different design principles. Tools achieving high precision can lack sensitivity in some applications. Conversely, tools with high sensitivity can suffer from low precision and require long computation time. In this paper, we present WEVOTE (WEighted VOting Taxonomic idEntification), a method that classifies metagenome shotgun sequencing DNA reads based on an ensemble of existing methods using k-mer-based, marker-based, and naive-similarity based approaches. Our evaluation on fourteen benchmarking datasets shows that WEVOTE improves the classification precision by reducing false positive annotations while preserving a high level of sensitivity. WEVOTE is an efficient and automated tool that combines multiple individual taxonomic identification methods to produce more precise and sensitive microbial profiles. WEVOTE is developed primarily to identify reads generated by MetaGenome Shotgun sequencing. It is expandable and has the potential to incorporate additional tools to produce a more accurate taxonomic profile. WEVOTE was implemented using C++ and shell scripting and is available at www.bitbucket.org/ametwally/wevote

LMethyR-SVM: Predict human enhancers using low methylated regions based on weighted support vector machines

Yang Dai — 2016-05-19

BackgroundThe identification of enhancer is a challenging task. Various types of epigenetic information including histone modification have been utilized in the construction of enhancer prediction models based on a diverse panel of machine learning models. However, DNA methylation profiles generated from the whole genome bisulfate sequencing (WGBS) have not been fully explored for their potential in enhancer prediction despite the fact that low methylated regions (LMRs) have been implied to be distal active regulatory regions.nnMethodIn this work we propose a prediction framework, LMethyR-SVM, using LMRs identified from cell-type-specific WGBS DNA methylation profiles based on an unlabeled-negative learning framework. In LMethyR-SVM, the set of cell-type-specific LMRs is further divided into three sets: reliable positive, like positive, and likely negative, according to their resemblance to a small set of experimentally validated enhancers in the VISTA database based on an estimated non-parametric density distribution. Then, the prediction model is trained by solving a weighted support vector machine.nnResultsWe demonstrate the performance of LMethyR-SVM by using the WGBS DNA methylation profiles derived from the H1 human embryonic stem cell type (H1) and the fetal lung fibroblast cell type (IMR90). The predicted enhancers are highly conserved with a reasonable validation rate based on a set of commonly used positive markers including transcription factors, p300 binding and DNase-I hypersensitive sites. In addition, we show evidence that the large fraction of LMethyR-SVM predicted enhancers are not predicted by ChromHMM in H1 cell type and they are more enriched for the FANTOM5 enhancers.nnConclusionOur work suggests that low methylated regions detected from the WGBS data are useful as complementary resources to histone modification marks in developing models for the prediction of cell type-specific enhancers.

Differential stimulation of the retina with glutamate toward a neurotransmitter-based retinal prosthesis

Corey Rountree — 2016-06-24

Subretinal stimulation of the retina with neurotransmitters, the normal means of conveying visual information, is a potentially better alternative to electrical stimulation widely used in current retinal prostheses for treating blindness from photoreceptor degenerative diseases. Yet, no retinal stimulation study exploiting the inner retinal pathways exists. Here, we demonstrate the feasibility of differentially stimulating retinal ganglion cells (RGCs) through the inner nuclear layer of the retina with glutamate, a primary neurotransmitter chemical, in a biomimetic way. We show that controlled pulsatile delivery of glutamate into the subsurface of explanted wild-type rat retinas elicits highly localized simultaneous inhibitory and excitatory spike rate responses in OFF and ON RGCs. We also present the spatiotemporal characteristics of RGC responses to subretinally injected glutamate and the therapeutic stimulation parameters. Our findings could pave the way for future development of a neurotransmitter-based subretinal prosthesis offering more naturalistic vision and better visual acuity than electrical prostheses.

The lexical categorization model: A computational model of left ventral occipito-temporal cortex activation in visual word recognition

Gagl, B. — 2016-11-03

To characterize the left-ventral occipito-temporal cortex (lvOT) role during reading in a quantitatively explicit and testable manner, we propose the lexical categorization model (LCM). The LCM assumes that lvOT optimizes linguistic processing by allowing fast meaning access when words are familiar and filter out orthographic strings without meaning. The LCM successfully simulates benchmark results from functional brain imaging. Empirically, using functional magnetic resonance imaging, we demonstrate that quantitative LCM simulations predict lvOT activation across three studies better than alternative models. Besides, we found that word-likeness, which is assumed as input to LCM, is represented posterior to lvOT. In contrast, a dichotomous word/non-word contrast, which is assumed as the LCMs output, could be localized to upstream frontal brain regions. Finally, we found that training lexical categorization results in more efficient reading. Thus, we propose a ventral-visual-stream processing framework for reading involving word-likeness extraction followed by lexical categorization, before meaning extraction.

Cell proliferation depends on the direct binding between PKM2 and AKAP-Lbc

Chen, X. — 2017-01-26

The M2 form of the glycolytic enzyme pyruvate kinase (PKM2) has generated much interest recently due to its important role in tumor metabolism. A yeast two-hybrid screen carried out by the Alliance for Cell Signaling suggests that PKM2 interacts with A-Kinase Anchoring Protein (AKAP)-Lbc.nnAKAP-Lbc (also known as AKAP13) is a scaffold protein that integrates signaling through multiple enzymes including protein kinases A and D and the small G protein Rho. AKAP-Lbc was originally identified in leukemic blast cells, and multiple reports implicate AKAP-Lbc in breast, prostate and thyroid cancers, however the role of AKAP-Lbc in cancer biology is not understood.nnCo-immunoprecipitation, pulldown and Bimolecular Fluorescence Complementation (BiFC) data indicate that PKM2 interacts with AKAP-Lbc. Mapping experiments indicate that PKM2 directly interacts with amino acid residues 1923-2817 of AKAP-Lbc. By disrupting the interaction between the two proteins with the expression of the AKAP-Lbc fragments, our data suggest that the binding between PKM2 and PKA plays a critical role in cell proliferation. The work indicates that the binding between AKAP-Lbc and PKM2 may be an important target to treat some cancers by reducing the cell proliferation.

Open Design 3D-Printable Adjustable Micropipette that meets ISO Standard for Accuracy

Brennan, M. — 2017-02-20

Scientific communities are drawn to the open source model as an increasingly utilitarian method to produce and share work. Initially used as a means to develop freely available software, open source projects have been applied to hardware including scientific tools. Increasing convenience of 3D printing has fueled the proliferation of open labware projects aiming to develop and share designs for scientific tools that can be produced in-house as cheap alternatives to commercial products. We present our design of a micropipette that is assembled from 3D-printable parts and some hardware that works by actuating a disposable syringe to a user adjustable limit. Graduations on the syringe are used to accurately adjust the set point to the desired volume. Our open design printed micropipette is assessed in comparison to a commercial pipette and meets ISO 8655 standards.

Reversed graph embedding resolves complex single-cell developmental trajectories

Qiu, X. — 2017-02-21

Organizing single cells along a developmental trajectory has emerged as a powerful tool for understanding how gene regulation governs cell fate decisions. However, learning the structure of complex single-cell trajectories with two or more branches remains a challenging computational problem. We present Monocle 2, which uses reversed graph embedding to reconstruct single-cell trajectories in a fully unsupervised manner. Monocle 2 learns an explicit principal graph to describe the data, greatly improving the robustness and accuracy of its trajectories compared to other algorithms. Monocle 2 uncovered a new, alternative cell fate in what we previously reported to be a linear trajectory for differentiating myoblasts. We also reconstruct branched trajectories for two studies of blood development, and show that loss of function mutations in key lineage transcription factors diverts cells to alternative branches on the a trajectory. Monocle 2 is thus a powerful tool for analyzing cell fate decisions with single-cell genomics.

Clarinet (CLA-1), a novel active zone protein required for synaptic vesicle clustering and release

Xuan, Z. — 2017-06-03

Active zone proteins cluster synaptic vesicles at presynaptic terminals and coordinate their release. In forward genetic screens we isolated a novel C. elegans active zone gene, clarinet (cla-1). cla-1 mutants exhibit defects in synaptic vesicle clustering, reduced spontaneous neurotransmitter release, increased synaptic depression and reduced synapse number. Ultrastructurally, cla-1 mutants have fewer synaptic vesicles adjacent to the dense projection and an increased number of docked vesicles. Cla-1 encodes 3 isoforms containing common C-terminal PDZ and C2 domains with homology to vertebrate active zone proteins Piccolo and RIM. The short isoform localizes exclusively to the active zone while a longer ~9000 amino acid isoform colocalizes with synaptic vesicles. Specific loss of CLA-1L results in synaptic vesicle clustering defects and increased synaptic depression, but not in reduced synapse number or mini frequency. Together our data indicate that specific isoforms of clarinet serve distinct functions, regulating synapse development, synaptic vesicle clustering and release.

Positional effects revealed in Illumina Methylation Array and the impact on analysis

Jiao, C. — 2017-06-22

With the evolution of rapid epigenetic research, Illumina Infinium HumanMethylation BeadChips have been widely used to study DNA methylation. However, in evaluating the accuracy of this method, we found that the commonly used Illumina HumanMethylation BeadChips are substantially affected by positional effects; the DNA samples location in a chip affects the measured methylation levels. We analyzed three HumanMethylation450 and three HumanMethylation27 datasets by using four methods to prove the existence of positional effects. Three datasets were analyzed further for technical replicate analysis or differential methylation CpG sites analysis. The pre- and post-correction comparisons indicate that the positional effects could alter the measured methylation values and downstream analysis results. Nevertheless, ComBat, linear regression and functional normalization could all be used to minimize such artifact. We recommend performing ComBat to correct positional effects followed by the correction of batch effects in data preprocessing as this procedure slightly outperforms the others. In addition, randomizing the sample placement should be a critical laboratory practice for using such experimental platforms. Code for our method is freely available at: https://github.com/ChuanJ/posibatch.

Common variants of NRXN1, LRP1B and RORA are associated with increased ventricular volumes in psychosis - GWAS findings from the B-SNIP deep phenotyping study

Alliey-Rodriguez, N. — 2017-08-11

Schizophrenia, Schizoaffective, and Bipolar Disorders share common illness traits, intermediate phenotypes and a partially overlapping polygenic basis. We performed GWAS on deep phenotyping data, including structural MRI and DTI, clinical, and behavioral scales from 1,115 cases and controls. Significant associations were observed with two cerebrospinal fluid volumes: the temporal horn of left lateral ventricle was associated with NRXN1, and the volume of the cavum septum pellucidum was associated with LRP1B and RORA. Both volumes were associated with illness. Suggestive associations were observed with local gyrification indices, fractional anisotropy and age at onset. The deep phenotyping approach allowed unexpected genetic sharing to be found between phenotypes, including temporal horn of left lateral ventricle and age at onset.

Minor spliceosome inactivation in the developing mouse cortex causes self-amplifying radial glial cell death and microcephaly.

Baumgartner, M. — 2017-08-31

Inactivation of the minor spliceosome has been linked to microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1). To interrogate how minor intron splicing regulates cortical development, we employed Emx1-Cre to ablate Rnu11, which encodes the minor spliceosome-specific U11 small nuclear RNA (snRNA), in the developing cortex (pallium). Rnu11 cKO mice were born with microcephaly, caused by death of self-amplifying radial glial cells (RGCs). However, both intermediate progenitor cells (IPCs) and neurons were produced in the U11-null pallium. RNAseq of the pallium revealed elevated minor intron retention in the mutant, particularly in genes regulating cell cycle. Moreover, the only downregulated minor intron-containing gene (MIG) was Spc24, which regulates kinetochore assembly. These findings were consistent with the observation of fewer RGCs entering cytokinesis prior to RGC loss, underscoring the requirement of minor splicing for cell cycle progression in RGCs. Overall, we provide a potential explanation of how disruption of minor splicing might cause microcephaly in MOPD1.nnSummary StatementHere we report the first mammalian model to investigate the role of the minor spliceosome in cortical development and microcephaly.nnList of abbreviations usedMOPD1=microcephalic osteodysplastic primordial dwarfism type 1; snRNA=small nuclear RNA; cKO=conditional knockout; NPC=neural progenitor cell; RGC=radial glial cell; IPC=intermediate progenitor cell; MIG=minor intron-containing gene

GeTFEP: a general transfer free energy profile for transmembrane proteins

Tian, W. — 2017-10-31

Free energy of transferring amino acid side-chains from aqueous environment into lipid bilayers, known as transfer free energy (TFE), provides important information on the thermodynamic stability of membrane proteins. In this study, we derived a TFE profile named General Transfer Free Energy Profile (GeTFEP) based on computation of the TFEs of 58 {beta}-barrel membrane proteins ({beta}MPs). The GeTFEP agrees well with experimentally measured and computationally derived TFEs. Analysis based on the GeTFEP shows that residues in different regions of the TM segments of {beta}MPs have different roles during the membrane insertion process. Results further reveal the importance of the sequence pattern of transmembrane strands in stabilizing {beta}MPs in the membrane environment. In addition, we show that GeTFEP can be used to predict the positioning and the orientation of {beta}MPs in the membrane. We also show that GeTFEP can be used to identify structurally or functionally important amino acid residue sites of {beta}MPs. Furthermore, the TM segments of -helical membrane proteins can be accurately predicted with GeTFEP, suggesting that the GeTFEP captures fundamental thermodynamic properties of amino acid residues inside membrane, and is of general applicability in studying membrane protein.

MeDEStrand: an improved method to infer genome-wide absolute methylation level from DNA enrichment experiment

Xu, J. — 2017-09-27

BackgroundDNA methylation of dinucleotide CpG is an essential epigenetic modification that plays a key role in transcription. Bisulfite conversion method is a "gold standard" for DNA methylation profiling that provides single nucleotide resolution. However, whole-genome bisulfite conversion is very expensive. Alternatively, DNA enrichment-based methods offer high coverage of methylated CpG dinucleotides with the lowest cost per CpG covered genome-wide and have been used widely. They measure the DNA enrichment of methyl-CpG binding, therefore do not directly provide absolute methylation levels. Further, the enrichment is influenced by confounding factors besides the methylation status, e.g., CpG density. Computational models that can accurately derive the absolute methylation levels from the enrichment data are necessary.nnResultsWe present MeDEStrand, a method uses sigmoid function to estimate and correct the CpG bias from the numbers of reads that fell within bins that divide the genome. In addition, unlike the previous methods, which estimate CpG bias based on reads mapped at the same genomic loci, MeDEStrand processes the reads for the positive and negative DNA strands separately. We compare the performance of MeDEStrand with three other state-of-the-art methods MEDIPS, BayMeth and QSEA on four independent datasets generated using immortalized cell lines (GM12878 and K562) and human patient primary cells (foreskin fibroblast and mammary epithelial). Based on the comparison between the inferred absolute methylation levels from MeDIP-seq and the corresponding RRBS data, MeDEStrand shows the best performance at high resolution of 25, 50 and 100 base pairs.nnConclusions MeDEStrand benefits from the estimation of CpG bias with a sigmoid function and the procedure to process reads mapped to the positive and negative DNA strands separately. MeDEStrand is a tool to infer whole-genome absolute DNA methylation level at the cost of enrichment-based methods with adequate accuracy and resolution. R package MeDEStrand and its tutorial is freely available for download at https://github.com/jxu1234/MeDEStrand.git

APOBEC3A Catalyzes Mutation and Drives Carcinogenesis In Vivo

Law, E. — 2019-12-27

The APOBEC3 family of antiviral DNA cytosine deaminases is implicated as the second largest source of mutation in cancer. This mutational process may be a causal driver or inconsequential passenger to the overall tumor phenotype. We show that human APOBEC3A expression in murine colon and liver tissues increases tumorigenesis. All other APOBEC3 family members including APOBEC3B did not as strongly promote liver tumor formation. DNA sequences from APOBEC3A-expressing animals display hallmark APOBEC signature mutations in TCA/T motifs. Bioinformatic comparisons of the observed APOBEC3A mutation signature in murine tumors, previously reported APOBEC3A and APOBEC3B mutation signatures in yeast, and reanalyzed APOBEC mutation signatures in human tumor data sets support cause-and-effect relationships for APOBEC3A-catalyzed deamination and mutagenesis in driving multiple human cancers.

Identifying strategies to target the metabolic flexibility of tumours.

Yuneva, M. — 2020-01-07

Plasticity of cancer metabolism can be a major obstacle for efficient targeting of tumour-specific metabolic vulnerabilities. Here, we identify and quantify the compensatory mechanisms following the inhibition of major pathways of central carbon metabolism in c-MYC-induced liver tumours. We find that glutaminase isoform Gls2, expressed in normal liver, compensates for the deletion of Gls1 isoform expressed in tumours. Inhibiting both glutaminases significantly delays tumourigenesis but does not completely block glutamine catabolism through the Krebs cycle. We reveal that glutamine catabolism is then driven by amidotransferases. Consistently, the synergistic effect of glutaminase and amidotransferase inhibitors on proliferation of mouse and human tumour cells is observed in vitro and in vivo. Furthermore, when Gls1 is deleted the Krebs cycle activity and tumour formation can also be significantly affected if glycolysis is co-inhibited (Gls1KO/Hk2KO). Finally, the inhibition of either serine (Psat1KO) or fatty acid (FasnKO) biosynthesis can be compensated by uptake of circulating nutrients. Thus, removing these nutrients from the diet produces synergistic effects on suppression of tumourigenesis. These results highlight the high flexibility of tumour metabolism and demonstrate how targeting compensatory mechanisms can improve a therapeutic outcome.

Cell autonomous versus systemic Akt isoform deletions uncovered new roles for Akt1 and Akt2 in breast cancer

Hay, N. — 2020-01-08

Studies in three mouse models of breast cancer identified profound discrepancies between cell autonomous and systemic Akt1 or Akt2 deletion on breast cancer tumorigenesis and metastasis. First, unlike systemic Akt1 deletion, which inhibits metastasis, cell autonomous Akt1 deletion does not. Second, systemic Akt2 deletion does not inhibit mammary tumorigenesis and metastasis, but cell autonomous Akt2 deletion eliminates ErbB2 expressing cells in the mammary gland and prevents tumorigenesis. However, the elevation in insulin by Akt2 systemic deletion hyperactivates tumor Akt, enabling ErbB2 expression, and exacerbates mammary tumorigenesis. Decreasing insulin level inhibits accelerated tumorigenesis by systemic Akt2 deletion. Single cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing the survival and mobilization of tumor-associated neutrophils. Importantly, neutrophil-specific deletion of Akt1 is sufficient to exert resistance to metastasis. The results underscore the importance of determining systemic effects rather than cell autonomous effects as a proof of concept for cancer therapy.

Salmonella Enteritidis Effector AvrA suppresses autophagy by reducing Beclin-1 protein

Jiao, Y. — 2020-01-14

Autophagy is a cellular process to clear pathogens. Salmonella enterica serovar Enteritidis (S.E) has emerged as one of the most important food-borne pathogens. However, major studies still focus on Salmonella enterica serovar Typhimurium. Here, we reported that AvrA, a S. Enteritidis effector, inhibited autophagy to promote bacterial survival in the host. We found that AvrA regulates the conversion of LC3 I into LC3 II and the enrichment of lysosomes. Beclin-1, a key molecular regulator of autophagy, was decreased after AvrA expressed strain colonization. In S.E-AvrA--infected cells, we found the increases of protein levels of p-JNK and p-c-Jun and the transcription level of AP-1. AvrA-reduction of Beclin-1 protein expression is through the JNK pathway. The JNK inhibitor abolished the AvrA-reduced Beclin-1 protein expression. Moreover, we identified that the AvrA mutation C186A abolished its regulation of Beclin-1 expression. In addition, AvrA protein interacted with Beclin-1. In organoids and infected mice, we explored the physiologically related effects and mechanism of AvrA in reducing Beclin-1 through the JNK pathway, thus attenuating autophagic responses. ImportanceSalmonella Enteritidis is an important pathogen with a public health concern and farm production risk, yet the host-pathogen interactions that govern the survival of S. Enteritidis infections are incompletely understood. Anti-bacterial autophagy provides potent cell-autonomous immunity against bacterial colonization. Here, we report that a new role for effector AvrA of S. Enteritidis in the reduction of Beclin-1 protein expression through the JNK pathway and the attenuation of the autophagic response in intestinal epithelial cells. This finding not only indicates an important role of S. Enteritidis effector in reducing host protein as a strategy to suppress autophagy, but also suggests manipulating autophagy as a new strategy to treat infectious diseases.

Potential depression and antidepressant-response biomarkers in human lymphoblast cell lines from treatment-responsive and treatment-resistant subjects: roles of SSRIs and omega-3 polyunsaturated fatty acids

Chukaew, P. — 2020-01-23

While several therapeutic strategies exist for depression, most antidepressant drugs require several weeks before reaching full biochemical efficacy and remission is not entirely achieved in many patients. Therefore, biomarkers for depression and drug-response would help tailor treatment strategies. This study made use of banked human lymphoblast cell lines (LCLs) from normal and depressed subjects; the latter divided into remitters and non-remitters. Due to the fact that previous studies have shown effects on growth factors, cytokines and elements of the cAMP generating system as potential biomarkers for depression and antidepressant action, these were examined in LCLs. Initial gene and protein expression profiles for signaling cascades related to neuroendocrine and inflammatory functions differ among the three groups. Growth factor genes, including VEGFA and BDNF were significantly down-regulated in cells from depressed subjects. In addition, omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported to act as both antidepressants and anti-inflammatories, but the mechanisms for these effects are not established. Here we showed that n-3 PUFAs and escitalopram (selective serotonin reuptake inhibitors, SSRIs) treatment increased adenylyl cyclase (AC) and BDNF gene expression in LCLs. These data are consistent with clinical observations showing that n-3 PUFA and SSRI have antidepressant affects, which may be additive. Contrary to observations made in neuronal and glial cells, n-3 PUFA treatment attenuated cAMP accumulation in LCLs. However, while lymphoblasts show paradoxical responses to neurons and glia, patient-derived lymphoblasts appear to carry potential depression biomarkers making them an important tool for studying precision medicine in depressive patients. Furthermore, these data validate usefulness of n-3 PUFAs in treatment for depression.

Membrane-associated α-tubulin is less acetylated in postmortem prefrontal cortex from depressed subjects relative to controls: cytoskeletal dynamics, HDAC6 and depression

Singh, H. — 2020-01-23

Cytoskeletal proteins and post-translational modifications play a role in mood disorders. Post-translational modifications of tubulin also alter microtubule dynamics. Furthermore, tubulin interacts closely with Gs, the G-protein responsible for activation of adenylyl cyclase. Postmortem tissue derived from depressed suicide brain showed increased Gs in lipid-raft domains compared to normal subjects. Gs, when ensconced in lipid-rafts, couples less effectively with adenylyl cyclase to produce cAMP and this is reversed by antidepressant treatment. A recent in-vitro study demonstrated that tubulin anchors Gs to lipid-rafts and that increased tubulin acetylation (due to HDAC-6 inhibition) and antidepressant treatment decreased the proportion of Gs complexed with tubulin. This suggested that deacetylated-tubulin might be more prevalent in depression. This study, examined tubulin acetylation in whole tissue homogenate, plasma-membrane and lipid-raft membrane domains in tissue from normal control (NC) subjects, depressed suicides and depressed non-suicides. While tissue homogenate showed no changes in [<]-tubulin/tubulin acetylation between control, depressed suicides and depressed non-suicides, plasma-membrane associated tubulin showed significant decreases in acetylation in depressed suicides and depressed non-suicides compared to controls. No change was seen in expression of the enzymes responsible for tubulin acetylation or deacetylation. These data suggest that during depression, membrane localized tubulin maintains a lower acetylation state, permitting increased sequestration of Gs in lipid-raft domains, where it is less likely to couple to adenylyl cyclase for cAMP production. Thus, membrane tubulin may play a role in mood disorders which could be exploited for diagnosis and treatment. Significance StatementThere is little understanding about the molecular mechanisms involved in the development of depression and in severe cases, suicide. Evidence for the role of microtubule modifications in progression of depressive disorders is emerging. These postmortem data provide strong evidence for membrane tubulin modification leading to reduced efficacy of the G protein, Gs, in depression. This study reveals a direct link between decreased tubulin acetylation in human depression and the increased localization of Gs in lipid-raft domains responsible for attenuated cAMP signaling. The evidence presented here suggest a novel diagnostic and therapeutic locus for depression.

N-3 polyunsaturated fatty acids promote astrocyte differentiation and neurotrophin production independent of cAMP in patient-derived neural stem cells

Yu, J.-Z. — 2020-01-23

Evidence from epidemiological and laboratory studies, as well as randomized placebo-controlled trials, suggests supplementation with n-3 polyunsaturated fatty acids (PUFAs) may be efficacious for treatment of major depressive disorder (MDD). The mechanisms underlying n-3 PUFAs potential therapeutic properties remain unknown. There are suggestions in the literature that glial hypofunction is associated with depressive symptoms and that antidepressants may normalize glial function. In this study, iPSC-derived neuronal stem cell lines were generated from individuals with MDD. Astrocytes differentiated from patient-derived neuronal stem cells (iNSCs) were verified by GFAP. Cells were treated with eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and stearic acid (SA). During astrocyte differentiation, we found that n-3 PUFAs increased GFAP expression and GFAP positive cell formation. BDNF and GDNF production were increased in the astrocytes derived from patients subsequent to n-3 PUFA treatment. Stearic Acid (SA) treatment did not have this effect. CREB activity (phosphorylated CREB) was also increased by DHA and EPA but not by SA. Furthermore, when these astrocytes were treated with n-3 PUFAs, the cAMP antagonist, RP-cAMPs did not block n- 3 PUFA CREB activation. However, the CREB specific inhibitor (666-15) diminished BDNF and GDNF production induced by n-3 PUFA, suggesting CREB dependence. Together, these results suggested that n-3 PUFAs facilitate astrocyte differentiation, and may mimic effects of some antidepressants by increasing production of neurotrophic factors. The CREB-dependence and cAMP independence of this process suggests a manner in which n-3 PUFA could augment antidepressant effects. These data also suggest a role for astrocytes in both MDD and antidepressant action.

A Tug of War Between Condensate Phases in a Minimal Macromolecular System

Ghosh, A. — 2020-01-30

Membraneless organelles formed via liquid-liquid phase separation (LLPS) contain a multitude of macromolecular species. A few of these species drive LLPS while most serve as regulators. The LLPS of SH35 (S) and PRM5 (P), two oppositely charged protein constructs, was promoted by a polyanion heparin (H) but suppressed by a cationic protein lysozyme (L). Here, using these four components alone, we demonstrate complex phase behaviors associated with membraneless organelles and uncover the underlying physical rules. The S:P, S:L, and P:H binaries form droplets, but the H:L binary forms precipitates, therefore setting off a tug of water between different phases within the S:P:H:L quaternary. We observe dissolution of precipitates upon compositional change, transformation from precipitates to droplet-like condensates over time, and segregation of S:L-rich and P:H-rich foci inside droplet-like condensates. A minimal macromolecular system can thus recapitulate membraneless organelles in essential ways and provide crucial physical understanding.

Primary Human Hepatocytes Maintain Long-term Functions in Porous Silk Scaffolds Containing Extracellular Matrix Proteins

Kukla, D. — 2020-01-31

The shortage of donor organs for transplantation has prompted the development of alternative implantable human liver tissues; however, the need for a clinically viable liver tissue that can be fabricated using physiologically-relevant primary human hepatocytes (PHHs) is unmet. Purified silk proteins provide desirable features for generating implantable tissues, such as sustainable sourcing from insects/arachnids, biocompatibility, tunable mechanical properties and degradation rates, and low immunogenicity upon implantation; however, the utility of such scaffolds to generate human liver tissues using PHHs remains unclear. Here, we show that the incorporation of type I collagen during the fabrication and/or autoclaving of silk scaffolds was necessary to enable robust PHH attachment/function. Scaffolds with small pores (73 +/- 25 {micro}m) promoted higher PHH functions than large pores (235 +/- 84 {micro}m). Further incorporation of growth-arrested 3T3-J2 fibroblasts into scaffolds enhanced PHH functions up to 5-fold for 5 months in culture, an unprecedented longevity, and functions were better retained than 2D configurations. Lastly, encapsulating PHHs within Matrigel while housed in the silk/collagen scaffold led to higher functions than Matrigel or silk/collagen alone. In conclusion, porous silk scaffolds are useful for generating long-term PHH +/- fibroblast tissues which may ultimately find applications in regenerative medicine and drug development.

Cell phone digital microscopy using an oil droplet

Szydlowski, N. A. — 2020-02-11

We introduce an accessible imaging method using droplets of microscope immersion and consumer-grade oils and a cell phone camera. We found that oil droplets were more resistant to evaporation than water droplets. We characterized the transverse magnification of oil and water droplets using cell phone screens and a resolution target. We further harnessed the close refractive index of cooking oils to that of the immersion oil and demonstrated their use as lenses for cell phone microscopy. Our method enables stable droplet-based optical imaging without specialized setups or manufacturing processes.

Vitamin D receptor protects against dysbiosis and tumorigenesis via the JAK/STAT pathway in intestine

Zhang, Y.-g. — 2020-02-19

BackgroundVitamin D exerts regulatory roles via vitamin D receptor (VDR) in mucosal immunity, host defense, and inflammation involving host factors and microbiome. Human Vdr gene variation shapes the microbiome and VDR deletion leads to dysbiosis. Low VDR expression and diminished vitamin D/VDR signaling are observed in colon cancer. Nevertheless, how intestinal epithelial VDR is involved in tumorigenesis through gut microbiota remains unknown. We hypothesized that intestinal VDR protects mice against dysbiosis via modulating the JAK/STAT pathway in tumorigenesis. To test our hypothesis, we used an azoxymethane/Dextran Sulfate Sodium-induced cancer model in intestinal VDR conditional knockout (VDR{Delta}IEC) mice, cell cultures, stem-cell derived colonoids, and human colon cancer samples. ResultsVDR{Delta}IEC mice have higher numbers of tumors with location shifted from distal to proximal colon. Fecal microbiota analysis showed that VDR deletion leads to bacterial profile shift from normal to susceptible carcinogenesis. We found enhanced bacterial staining in mouse and human tumors. Microbial metabolites from VDR{Delta}IEC mice showed elevated secondary bile acids, consistent with the observations in human CRC. We further identified that VDR protein bound to the Jak2 promoter, suggesting that VDR transcriptionally regulated Jak2. The JAK/STAT pathway is critical in intestinal and microbial homeostasis. Fecal samples from VDR{Delta}IEC mice activate the STAT3 activation in human and mouse organoids. Lack of VDR led to hyperfunction of Jak2 in respond to intestinal dysbiosis. A JAK/STAT inhibitor abolished the microbiome-induced activation of STAT3. ConclusionWe provide insights into the mechanism of VDR dysfunction leading to dysbiosis and tumorigenesis. It indicates a new target -- microbiome and VDR for prevention of cancer.

Transcriptional control of parallel-acting pathways that remove discrete presynaptic proteins in remodeling neurons

Miller, D. M. — 2020-02-24

Synapses are actively dismantled to mediate circuit refinement, but the developmental pathways that regulate synaptic disassembly are largely unknown. We have previously shown that the epithelial sodium channel UNC-8 triggers an activity-dependent mechanism that drives the removal of presynaptic proteins liprin-/SYD-2, Synaptobrevin/SNB-1, RAB-3 and Endophilin/UNC-57 in remodeling GABAergic neurons in C. elegans (Miller-Fleming et al., 2016). Here, we report that the transcription factor Iroquois/IRX-1 regulates UNC-8 expression as well as an additional pathway, independent of UNC-8, that functions in parallel to dismantle functional presynaptic terminals. We show that the additional IRX-1-regulated pathway is selectively required for the removal of the presynaptic proteins, Munc13/UNC-13 and ELKS, which normally mediate synaptic vesicle fusion and neurotransmitter release. Our findings are notable because they highlight the key role of transcriptional regulation in synapse elimination and reveal parallel-acting pathways that orchestrate synaptic disassembly by removing specific active zone proteins.

BoneMA - Synthesis and Characterization of a Methacrylated Bone-derived Hydrogel for Bioprinting of Vascularized Tissues

S., P. P. — 2020-03-04

It has long been proposed that recapitulating the extracellular matrix (ECM) of native human tissues in the laboratory may enhance the regenerative capacity of engineered scaffolds in-vivo. Organ- and tissue-derived decellularized ECM biomaterials have been widely used for tissue repair, especially due to their intrinsic biochemical cues that can facilitate repair and regeneration. The main purpose of this study was to synthesize a new photocrosslinkable human bone-derived ECM hydrogel for bioprinting of vascularized scaffolds. To that end, we demineralized and decellularized human bone fragments to obtain a bone matrix, which was further processed and functionalized with methacrylate groups to form a photocrosslinkable methacrylate bone ECM hydrogel - BoneMA. The mechanical properties of BoneMA were tunable, with the elastic modulus increasing as a function of photocrosslinking time, while still retaining the nanoscale features of the polymer networks. The intrinsic cell-compatibility of the bone matrix ensured the synthesis of a highly cytocompatible hydrogel. The bioprinted BoneMA scaffolds supported vascularization of endothelial cells and within a day led to the formation of interconnected vascular networks. We propose that such a quick vascular network formation was due to the host of pro-angiogenic biomolecules present in the bone ECM matrix. Further, we also demonstrate the bioprintability of BoneMA in microdimensions as injectable ECM-based building blocks for microscale tissue engineering in a minimally invasive manner. We conclude that BoneMA may be a useful hydrogel system for tissue engineering and regenerative medicine.

Out-of-register parallel β-sheets and antiparallel β-sheets coexist in 150 kDa oligomers formed by Aβ(1-42)

Gao, Y. — 2020-03-04

We present solid-state NMR measurements of {beta}-strand secondary structure and inter-strand organization within a 150 kDa oligomeric aggregate of the 42-residue variant of the Alzheimers amyloid-{beta} peptide (A{beta}(1-42)). This oligomer is characterized by a structure that cannot be explained by any previously proposed model for aggregated A{beta}. We build upon our previous report of a {beta}-strand spanned by residues 30-42, which arranges into an antiparallel {beta}-sheet. New results presented here indicate that there is a second {beta}-strand formed by residues 11-24. We show negative results for NMR experiments designed to reveal antiparallel {beta}-sheets formed by this {beta}-strand. Remarkably, we show that this strand is organized into a parallel {beta}-sheet despite the co-existence of an antiparallel {beta}-sheet in the same structure. In addition, the in-register parallel {beta}-sheet commonly observed for amyloid fibril structure does not apply to residues 11-24 in the 150 kDa oligomer. Rather, we present evidence for an inter-strand registry shift of 3 residues that alternates in direction between adjacent molecules along the {beta}-sheet. We corroborated this unexpected scheme for {beta}-strand organization using multiple 2-dimensional NMR and 13C-13C dipolar recoupling experiments. Our findings indicate a previously unknown assembly pathway and inspire a suggestion as to why this aggregate does not grow to larger sizes.

Single-chain lanthanide luminescence biosensors for cell-based imaging and screening of protein-protein interactions

Chen, T. — 2020-03-11

Research tools that enable imaging or analysis of protein-protein interactions (PPIs) directly within living cells provide unique and valuable biological insights and can also aid drug discovery efforts. Here, we present lanthanide-based, Forster resonance energy transfer (lanthanide-based FRET, or LRET) biosensors for time-gated luminescence (TGL) imaging or multiwell plate analysis of PPIs. Polypeptide chains comprised of an alpha helical linker flanked by a Tb(III) complex, GFP and two binding domains exhibit large differences in long-lifetime, Tb(III)-to-GFP LRET-sensitized emission between open (unbound) and closed (bound) states. We used TGL microscopy to image ca. 500% increases in Tb(III)-to-GFP LRET following rapamycin addition to NIH 3T3 cells that expressed biosensors bearing FKBP12 and the rapamycin binding domain of m-Tor (FRB) at each terminus. Much larger signal changes, up to ca. 2500%, were observed when cells were grown in 96-well or 384-well plates and analyzed using a TGL plate reader. We also measured the interaction of p53 and HDM2 and its inhibition within intact HeLa cells grown in 96-well plates and estimated a z-factor of 0.5 for the assay. The modular design and high dynamic range of Tb(III)-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

Establishing cell-intrinsic limitations in cell cycle progression controls graft growth and promotes differentiation of pancreatic endocrine cells

Sui, L. — 2020-03-14

Limitations in cell proliferation are a key barrier to reprogramming differentiated cells to pluripotent stem cells, and conversely, acquiring these limitations may be important to establish the differentiated state. The pancreas, and beta cells in particular have a low proliferative potential, which limits regeneration, but how these limitations are established is largely unknown. Understanding proliferation potential is important for the safty of cell replacement therapy with cell products made from pluripotent stem cell which have unlimited proliferative potential. Here we test a novel hypothesis, that these limitations are established through limitations in S-phase progression. We used a stem cell-based system to expose differentiating stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest, impairing S-phase entry, or S-phase completion. Upon release from these molecules, we determined growth potential, differentiation and function of insulin-producing endocrine cells both in vitro and after grafting in vivo. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells towards insulin-producing cells, improved the stability of the differentiated state, and protected mice from diabetes without the formation of cystic growths. Therefore, a compromised ability to enter S-phase and replicate the genome is a functionally important property of pancreatic endocrine differentiation, and can be exploited to generate insulin-producing organoids with predictable growth potential after transplantation.

Membrane Association and Functional Mechanism of Synaptotagmin-1 in Triggering Vesicle Fusion

Prasad, R. — 2020-03-14

Upon Ca2+ influx, synaptic vesicles fuse with the presynaptic plasma membrane (PM) to release neurotransmitters. Membrane fusion is triggered by synaptotagmin-1, a transmembrane protein in the vesicle membrane (VM), but the mechanism is under debate. Synaptotagmin-1 contains a single transmembrane helix (TM) and two tandem C2-domains (C2A and C2B). The present study aimed to use molecular dynamics simulations to elucidate how Ca2+-bound synaptotagmin-1, by simultaneously associating with VM and PM, brings them together for fusion. While C2A stably associates with VM via two Ca2+-binding loops, C2B has a propensity to partially dissociate. Importantly, an acidic motif in the TM-C2A linker competes with VM for interacting with C2B, thereby flipping its orientation to face PM. Subsequently C2B can readily associate with PM via a polybasic cluster and a Ca2+-binding loop. These results delineate the functional process of fusion triggered by synaptotagmin-1.

Autocatalytic-Protection for an Unknown Locus CRISPR-Cas Countermeasure for Undesired Mutagenic Chain Reactions

Schonfeld, E. — 2020-03-25

The mutagenic chain reaction (MCR) is a genetic tool to use a CRISPR-Cas construct to introduce a homing endonuclease, allowing gene drive to influence whole populations in a minimal number of generations1,2,3. The question arises: if an active genetic terror event is released into a population, could we prevent the total spread of the undesired allele4? Thus far, MCR protection methods require knowledge of the terror locus5. Here we introduce a novel approach, an autocatalytic-Protection for an Unknown Locus (a-PUL), whose aim is to spread through a population and arrest and decrease an active terror events spread without any prior knowledge of the terror-modified locus, thus allowing later natural selection and ERACR drives to restore the normal locus6. a-PUL, using a mutagenic chain reaction, includes (i) a segment encoding a non-Cas9 endonuclease capable of homology-directed repair suggested as Type II endonuclease Cpf1 (Cas12a), (ii) a ubiquitously-expressed gene encoding a gRNA (gRNA1) with a U4AU4 3'-overhang specific to Cpf1 and with crRNA specific to some desired genomic sequence of non-coding DNA, (iii) a ubiquitously-expressed gene encoding two gRNAs (gRNA2/gRNA3) both with tracrRNA specific to Cas9 and crRNA specific to two distinct sites of the Cas9 locus, and (iv) homology arms flanking the Cpf1/gRNA1/gRNA2/gRNA3 cassette that are identical to the region surrounding the target cut directed by gRNA17. We demonstrate the proof-of-concept and efficacy of our protection construct through a Graphical Markov model and computer simulation.

Brainstem development requires galactosylceramidase and is critical for the pathogenesis of Krabbe Disease

Weinstock, N. I. — 2020-03-25

Krabbe disease (KD) is caused by a deficiency of galactosylceramidase (GALC), which induces demyelination and neurodegeneration due to accumulation of cytotoxic psychosine. Hematopoietic stem cell transplantation (HSCT) improves clinical outcomes in KD patients only if delivered pre-symptomatically. We hypothesized that the restricted temporal efficacy of HSCT reflects a requirement for GALC in early brain development. Using a novel Galc floxed allele, we induced ubiquitous GALC ablation (Galc-iKO) at various postnatal timepoints and identified a critical period of vulnerability to GALC ablation between P4-6. Early Galc-iKO induction caused a worse KD phenotype, higher psychosine levels, and a significantly shorter life-span. Intriguingly, GALC expression peaks during this critical developmental period. Further analysis revealed a novel cell autonomous role for GALC in the development and maturation of immature T-box-brain-1 positive brainstem neurons. These data identify a perinatal developmental period, in which neuronal GALC expression influences brainstem development that is critical for KD pathogenesis.

The hexosamine biosynthesis pathway is a targetable liability in lung cancers with concurrent KRAS and LKB1 mutations.

Kim, J. — 2020-04-14

In non-small cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and the tumor suppressor STK11 encoding the kinase LKB1 result in aggressive tumors prone to metastasis but with liabilities arising from reprogrammed metabolism. We previously demonstrated perturbed nitrogen metabolism and addiction to an unconventional pathway of pyrimidine synthesis in KRAS/LKB1 co-mutant (KL) cancer cells. To gain broader insight into metabolic reprogramming in NSCLC, we analyzed tumor metabolomes in a series of genetically engineered mouse models with oncogenic KRAS combined with mutations in LKB1 or p53. Metabolomics and gene expression profiling pointed towards an activation of the hexosamine biosynthesis pathway (HBP), another nitrogen-related metabolic pathway, in both mouse and human KL mutant tumors. KL cells contain high levels of HBP metabolites, higher flux through the HBP pathway and elevated dependence on the HBP enzyme Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2). GFPT2 inhibition selectively reduced KL cell growth in culture and xenografts. Our results define a new metabolic vulnerability in KL tumors and provide a rationale for targeting GFPT2 in this aggressive NSCLC subtype.

Neuronal complexity is attenuated in chronic migraine and restored by HDAC6 inhibition

Bertels, Z. — 2020-04-22

Migraine is the third most prevalent disease worldwide but the mechanisms that underlie migraine chronicity are poorly understood. Cytoskeletal flexibility is fundamental to neuronal-plasticity and is dependent on dynamic microtubules. Histone-deacetylase-6 (HDAC6) decreases microtubule dynamics by deacetylating its primary substrate, -tubulin. We use validated models of migraine to show that HDAC6-inhibition is a promising migraine treatment and reveal an undiscovered cytoarchitectural basis for migraine chronicity. The human migraine trigger, nitroglycerin, produced chronic migraine-associated pain and decreased neurite growth in headache-processing regions, which were reversed by HDAC6 inhibition. Cortical spreading depression (CSD), a physiological correlate of migraine aura, also decreased cortical neurite growth, while HDAC6-inhibitor restored neuronal complexity and decreased CSD. Importantly, a calcitonin gene-related peptide receptor antagonist also restored blunted neuronal complexity induced by nitroglycerin. Our results demonstrate that disruptions in neuronal cytoarchitecture are a feature of chronic migraine, and effective migraine therapies might include agents that restore microtubule/neuronal plasticity.

Redundant cytokine requirement for intestinal microbiota-induced Th17 cell differentiation in draining lymph nodes

Sano, T. — 2020-04-22

Differentiation of intestinal T helper 17 (Th17) cells, which contribute to mucosal barrier protection from invasive pathogens, is dependent on colonization with distinct commensal bacteria. Segmented filamentous bacteria (SFB) are sufficient to support Th17 cell differentiation in mouse, but the molecular and cellular requirements for this process remain incompletely characterized. Here we show that intestine-draining mesenteric lymph nodes (MLN) are the dominant site of SFB-induced intestinal Th17 cell differentiation. Subsequent migration of these cells to the intestinal lamina propria is dependent on their up-regulation of integrin {beta}7. Stat3-dependent induction of ROR{gamma}t, the Th17 cell-specifying transcription factor, largely depends on IL-6, but signaling through the receptors for IL-21 and IL-23 can compensate for absence of IL-6 to promote SFB-directed Th17 cell differentiation. These results indicate that redundant cytokine signals guide commensal microbe-dependent Th17 cell differentiation in the MLN and accumulation of the cells in the lamina propria.

Impairment of the Gβγ-SNAP25 brake on exocytosis enhances insulin action, protects against diet-induced obesity, and promotes adipocyte browning

Ceddia, R. P. — 2020-04-30

Negative regulation of exocytosis from secretory cells throughout the body is accomplished through inhibitory signals from Gi/o G protein-coupled receptors by G{beta}{gamma} subunit inhibition of two common mechanisms: (i) decreased calcium entry and (ii) direct interaction of G{beta}{gamma} with the Soluble N-ethylmaleimide-sensitive factor Attachment Protein (SNAP) Receptor (SNARE) plasma membrane fusion machinery. We have previously shown that disabling the second mechanism with a truncation of SNAP25 (SNAP25{Delta}3/{Delta}3) decreases the affinity of G{beta}{gamma} for the SNARE complex, leaving exocytotic fusion as well as modulation of calcium entry intact but disabling GPCR inhibition of exocytosis. Here we report significant beneficial metabolic remodeling in mice carrying this mutation. Chow-fed SNAP25{Delta}3/{Delta}3 mice exhibit enhanced insulin sensitivity and increased beiging of white fat. In response to a high fat diet, the metabolic protection was amplified in SNAP25{Delta}3/{Delta}3 mice. Glucose homeostasis, whole body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. This metabolic protection in SNAP25{Delta}3/{Delta}3 mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting basal autonomic activity is required. Direct electrode stimulation of sympathetic neurons exocytosis from SNAP25{Delta}3/{Delta}3 inguinal adipose depot resulted in enhanced and prolonged norepinephrine release. Thus, the G{beta}{gamma}-SNARE interaction represents a cellular mechanism that deserves further exploration as a new avenue for combatting metabolic disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/069138v4_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@2c1fa0org.highwire.dtl.DTLVardef@d076ceorg.highwire.dtl.DTLVardef@27e351org.highwire.dtl.DTLVardef@bce219_HPS_FORMAT_FIGEXP M_FIG C_FIG

Structural basis of Rgg protein binding to their regulatory pheromones and target DNA promoters

Capodagli, G. C. — 2020-04-30

Rgg family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in Streptococcus species, controlling virulence, antimicrobial resistance, and biofilm formation. Rgg2 and Rgg3 function is regulated by their interaction with oligopeptide quorum-sensing signals called short hydrophobic peptides (SHPs). The molecular basis of Rgg-SHP and Rgg-target DNA promoter specificity was unknown. To close this gap, we determined the cryo-EM structure of Streptococcus thermophilus Rgg3 bound to its quorum-sensing signal, SHP3, and the X-ray crystal structure of Rgg3 alone. Comparison of these structures to that of an Rgg in complex with cyclosporin A (CsA), an inhibitor of SHP-induced Rgg activity, reveals the molecular basis of CsA function. Furthermore, to determine how Rgg proteins recognize DNA promoters, we determined X-ray crystal structures of both S. dysgalactiae Rgg2 and S. thermophilus Rgg3 in complex with their target DNA promoters. The physiological importance of the observed Rgg-DNA interactions was dissected using in vivo genetic experiments and in vitro biochemical assays. Based on these structure-function studies, we present a revised unifying model of Rgg regulatory interplay. In contrast to existing models, where Rgg2 proteins are transcriptional activators and Rgg3 proteins are transcriptional repressors, we propose that both are capable of transcriptional activation. However, when Rgg proteins with different activation requirements compete for the same DNA promoters, those with more stringent activation requirements function as repressors by blocking promoter access of the SHP-bound conformationally active Rgg proteins. While a similar gene expression regulatory scenario has not been previously described, in all likelihood it is not unique to streptococci. Significance StatementSecreted peptide pheromones regulate critical biological processes in Gram-positive bacteria. In streptococci such as the human pathogen S. pyogenes, oligopeptide pheromones, like the short hydrophobic peptides (SHPs), regulate virulence, antimicrobial resistance, and biofilm formation. SHPs directly regulate the activity of transcription factors called Rgg2 and Rgg3. We present the cryo-EM structure of Rgg3 in complex with SHP3, as well as X-ray crystal structures of Rgg2 bound to target promoter DNA, Rgg3 bound to target promoter DNA, and Rgg3 alone. Based on the cryo-EM, X-ray crystallographic, biochemical, and genetic studies presented here, we provide not only detailed mechanistic insight into the molecular basis of Rgg3-SHP3, Rgg2-DNA, and Rgg3-DNA binding specificity, but also a new model of transcription factor regulatory interplay.

Trapped Topoisomerase II initiates formation of de novo duplications via the nonhomologous end-joining pathway in yeast

Stantial, N. — 2020-05-04

Topoisomerase II (Top2) is an essential enzyme that resolves catenanes between sister chromatids as well as supercoils associated with the over- or under-winding of duplex DNA. Top2 alters DNA topology by making a double-strand break (DSB) in DNA and passing an intact duplex through the break. Each component monomer of the Top2 homodimer nicks one of the DNA strands and forms a covalent phosphotyrosyl bond with the 5 end. Stabilization of this intermediate by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. We describe the isolation of a yeast top2 mutant (top2- F1025Y,R1128G) whose product generates a stabilized cleavage intermediate in vitro. In yeast cells, overexpression of the top2- F1025Y,R1128G allele is associated with a novel mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Top2-associated duplications are promoted by the clean removal of the enzyme from DNA ends and are suppressed when the protein is removed as part of an oligonucleotide. TOP2 cells treated with etoposide exhibit the same mutation signature, as do cells that over-express the wild-type protein. These results have implications for genome evolution and are relevant to the clinical use of chemotherapeutic drugs that target Top2. SIGNIFICANCE STATEMENTDNA-strand separation during transcription and replication creates topological problems that are resolved by topoisomerases. These enzymes nick DNA strands to allow strand passage and then reseal the broken DNA to restore its integrity. Topoisomerase II (Top2) nicks complementary DNA strands to create double-strand break (DSBs) intermediates that can be stabilized by chemotherapeutic drugs and are toxic if not repaired. We identified a mutant form of yeast Top2 that forms stabilized cleavage intermediates in the absence of drugs. Over- expression of the mutant Top2 was associated with a unique mutation signature in which small (1-4 bp), unique segments of DNA were duplicated. These de novo duplications required the nonhomologous end-joining pathway of DSB repair, and their Top2-dependence has clinical and evolutionary implications.

Meta-Signer: Metagenomic Signature Identifier based on Rank Aggregation of Features

Reiman, D. — 2020-05-10

BackgroundThe advance of metagenomic studies provides the opportunity to identify microbial taxa that are associated to human diseases. Multiple methods exist for the association analysis. However, the results could be inconsistent, presenting challenges in interpreting the host-microbiome interactions. To address this issue, we introduce Meta-Signer, a novel Metagenomic Signature Identifier tool based on rank aggregation of features identified from multiple machine learning models including Random Forest, Support Vector Machines, LASSO, Multi-Layer Perceptron Neural Networks, and our recently developed Convolutional Neural Network framework (PopPhy-CNN). Meta-Signer generates ranked taxa lists by training individual machine learning models over multiple training partitions and aggregates them into a single ranked list by an optimization procedure to represent the most informative and robust microbial features. Meta-Signer can rank taxa using two input forms of the data: the relative abundances of the original taxa and taxa from the populated taxonomic trees generated from the original taxa. The latter form allows the evaluation of the association of microbial features at different taxonomic levels to the disease, which is attributed to our novel model of PopPhy-CNN. ResultsWe evaluate Mega-Signer on five different human gut-microbiome datasets. We demonstrate that the features derived from Meta-Signer were more informative compared to those obtained from other available feature ranking methods. The highly ranked features are strongly supported by published literature. ConclusionMeta-Signer is capable of deriving a robust set of microbial features at multiple taxonomic levels for the prediction of host phenotype. Meta-Signer is user-friendly and customizable, allowing users to explore their datasets quickly and efficiently.

rest2vec: Vectorizing the resting-state functional connectome using graph embedding

Morrissey, Z. D. — 2020-05-12

Resting-state functional magnetic resonance imaging (O_SCPLOWRSC_SCPLOWO_SCPCAP-C_SCPCAPO_SCPLOWFMRIC_SCPLOW) is widely used in connectomics for studying the functional relationships between regions of the human brain. O_SCPLOWRSC_SCPLOWO_SCPCAP-C_SCPCAPO_SCPLOWFMRIC_SCPLOW connectomics, however, has inherent analytical challenges, such as accounting for negative correlations. In addition, functional relationships between brain regions do not necessarily correspond to their anatomical distance, making the intrinsic geometry of the functional connectome less well understood. Recent techniques in natural language processing and machine learning, such as word2vec, have used embedding methods to map high-dimensional data into meaningful vector spaces. Inspired by this approach, we have developed a graph embedding pipeline, rest2vec, for studying the intrinsic geometry of functional connectomes. We demonstrate how rest2vec uses the phase angle spatial embedding (O_SCPLOWPHASEC_SCPLOW) method with dimensionality reduction techniques to embed the functional connectome into lower dimensions. Rest2vec can also be linked to the maximum mean discrepancy (O_SCPLOWMMDC_SCPLOW) metric to assign functional modules of the connectome in a continuous manner, improving upon traditional binary classification methods. Together, this allows for studying the functional connectome such that the full range of correlative information is preserved and gives a more informed understanding of the functional organization of the brain.

CTLA-4 blockade reverses the Foxp3+ T-regulatory-cell suppression of anti-tuberculosis T-cell effector responses

Shao, L. — 2020-05-13

BackgroundsIt has been well described that Foxp3+ T regulatory (Treg) cells suppress immune responses and that murine cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) can control the function of Foxp3+Treg cells. However, it remains unknown about the role of CTLA-4 pathway in Treg suppression of T cell responses in tuberculosis (TB). MethodsWe assessed TB-driven changes in CTLA-4-expressing Foxp3+ Treg and conducted CTLA-4 blocking mechanistic studies ex vivo in 126 subjects with active TB, latent TB or uninfected statuses. ResultsFrequencies of CTLA-4-expressing Treg cells were increased in the circulation of pulmonary TB patients and in the pleural compartment of TB pleuritis. Six-month anti-TB treatment significantly reduced CTLA-4+ Treg subset. Notably, antibody blocking of CTLA-4 pathway (CTLA-4 blockade) reversed the ability of Treg cells to suppress anti-TB Th1 responses and abrogated the Treg-mediated suppression of TB antigen-stimulated proliferative response. The CTLA-4 blockade reversed the Treg suppression of the ability of T cells to restrict intracellular BCG and M. tuberculosis growth in macrophages. InterpretationThe study uncovered previously-unreported observations implicating that the CTLA-4 blockade abrogates the capability of Treg cells to suppress anti-TB immune responses or immunity. Findings support the rationale for exploring the CTLA-4 blockade as potential host-directed therapy against TB. FundThis work is supported in part by the National Natural Science Foundation of China (30901277, 81671553, 81501359), and the Key Technologies Research and Development Program for Infectious Diseases of China (2017ZX10201302-004).

Exosomes Facilitate Transmission of SARS-CoV-2 Genome into Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Kwon, Y. — 2020-05-14

ABSTRACTThe novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into a worldwide pandemic. Early data suggest that the prevalence and severity of COVID-19 appear to be higher among patients with underlying cardiovascular risk factors. Despite the expression of angiotensin-converting enzyme 2 (ACE2), a functional receptor for SARS-CoV-2 infection, in cardiomyocytes, there has been no conclusive evidence of direct viral infection although the presence of inflammation and viral genome within the hearts of COVID-19 patients have been reported. Here we transduced A549 lung epithelial cells with lentivirus overexpressing selected genes of the SARS-CoV-2. We then isolated extracellular vesicles (EVs) from the supernatant of A549 cells and detected the presence of viral RNA within the purified EVs. Importantly, we observed that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were able to actively uptake these EVs and viral genes were subsequently detected in the cardiomyocytes. Accordingly, uptake of EVs containing viral genes led to an upregulation of inflammation-related genes in hiPSC-CMs. Thus, our findings indicate that SARS-CoV-2 RNA-containing EVs represent an indirect route of viral RNA entry into cardiomyocytes.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Genome-wide effects of the antimicrobial peptide apidaecin on translation termination

Vazquez-Laslop, N. — 2020-05-20

Biochemical studies suggested that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Here, genome-wide analysis revealed that Api arrests translating ribosomes at stop codons and causes pronounced queuing of the trailing ribosomes. By sequestering the available release factors, Api promotes pervasive stop codon bypass, leading to expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate development of new medicines and research tools for genome exploration.

Using Conditional Generative Adversarial Networks to Boost the Performance of Machine Learning in Microbiome Datasets

Reiman, D. — 2020-05-21

The microbiome of the human body has been shown to have profound effects on physiological regulation and disease pathogenesis. However, association analysis based on statistical modeling of microbiome data has continued to be a challenge due to inherent noise, complexity of the data, and high cost of collecting large number of samples. To address this challenge, we employed a deep learning framework to construct a data-driven simulation of microbiome data using a conditional generative adversarial network. Conditional generative adversarial networks train two models against each other while leveraging side information learn from a given dataset to compute larger simulated datasets that are representative of the original dataset. In our study, we used a cohorts of patients with inflammatory bowel disease to show that not only can the generative adversarial network generate samples representative of the original data based on multiple diversity metrics, but also that training machine learning models on the synthetic samples can improve disease prediction through data augmentation. In addition, we also show that the synthetic samples generated by this cohort can boost disease prediction of a different external cohort.

Proteomic Profiling of the Human Dentin Identifies Age-Related Differences in the Composition and Solubility of the Matrisome

Reis, M. C. — 2020-05-28

ObjectiveDevise a pipeline to investigate the protein composition of the human root dentin extracellular matrix (ECM) from single individuals of different age cohorts. DesignIndividual cervical root dentin of sound human molars from two age brackets, young (18-25 years old; n=3) and old (75-85 years old; n=3), were cut and pulverized. Protein extraction and fractionation were completed by sequential demineralization with EDTA buffer, chaotropic extraction with guanidine hydrochloride, and urea. The resulting protein extracts of differential solubility were digested into peptides and peptides were analyzed by mass spectrometry. Data generated for this study are available via ProteomeXchange, identifier PXD018320. ResultsWe found that protein extracts of different solubilities present distinct biochemical compositions. We further define the matrisome of young (48 proteins) and old (50 proteins) human root dentin and report the identification of compositional and structural differences in ECM proteins from young and old teeth. ConclusionOur study provides a rigorous pipeline, from sample preparation to data analysis, to investigate the ECM composition - or matrisome - of the dentin. This pipeline has the potential to lead to the discovery of biomarkers of tooth aging and health.

Molecular Basis of Far-red Sensing in Cyanobacteriochrome

Bandara, S. — 2020-06-03

Cyanobacteriochromes are small, panchromatic photoreceptors in the phytochrome superfamily that regulate diverse light-mediated adaptive processes in cyanobacteria. The molecular basis of far-red (FR) light perception by cyanobacteriochromes is currently unknown. Here we report the crystal structure of a far-red-sensing cyanobacteriochrome from Anabaena cylindrica PCC 7122, which exhibits a reversible far-red/orange photocycle. The 2.7 [A] structure of its FR-absorbing dark state, determined by room temperature serial crystallography and cryo-crystallography, reveals an all-Z,syn configuration of its bound linear tetrapyrrole (bilin) chromophore that is less extended than the bilin chromophores of all known phytochromes. Based on structural comparisons with other bilin-binding proteins and extensive spectral analyses on mutants, we identify key protein-chromophore interactions that enable far-red sensing in bilin-binding proteins. We propose that FR-CBCRs employ two distinct tuning mechanisms, which work together to produce a large batho-chromatic shift. Findings of this work have important implications for development and improvement of photoproteins with far-red absorption and fluorescence. Significance StatementPhytochromes are well known far-red-light sensors found in plants that trigger adaptive responses to facilitate competition for light capture with neighboring plants. Red- and far-red-sensing are critical to cyanobacteria living in the far-red-enriched shade of plants. Here we report the crystal structure of a far-red-sensing cyanobacteriochrome, a distant cyanobacterial relative of phytochrome. These studies shed insight into the poorly understood molecular basis of far-red-sensing by phytobilin-based photoreceptors. Owing to the deep tissue penetration of far-red light, far-red-sensing photoreceptors offer promising protein scaffolds for developing gene-based photoswitches, optoacoustic contrast agents and fluorescent probes for in situ imaging and optogenetic applications.

Ultra-structural analysis and morphological changes during the differentiation of trophozoite to cyst in Entamoeba invadens

Singh, N. — 2020-06-03

Entamoeba Histolytica, a pathogenic parasite, is the causative organism of amoebiasis and uses human colon to complete its life cycle. It destroys intestinal tissue leading to invasive disease. Since it does not form cyst in culture medium, a reptilian parasite Entamoeba invadens serves as the model system to study encystation. Detailed investigation on the mechanism of cyst formation, information on ultra-structural changes and cyst wall formation during encystation are still lacking in E. invadens. Here, we used electron microscopy to study the ultrastructural changes during cyst formation and showed that the increase in heterochromatin patches and deformation of nuclear shape were early events in encystation. These changes peaked at ~20h post induction, and normal nuclear morphology was restored by 72h. Two types of cellular structures were visible by 16h. One was densely stained and consisted of the cytoplasmic mass with clearly visible nucleus. The other consisted of membranous shells with large vacuoles and scant cytoplasm. The former structure developed into the mature cyst while the latter structure was lost after 20h, This study of ultra-structural changes during encystation in E. invadens opens up the possibilities for further investigation into the mechanisms involved in this novel process.

The conserved ASCL1/MASH-1 ortholog HLH-3 specifies sex-specific ventral cord motor neuron fate in C. elegans

Perez, L. M. — 2020-06-05

Neural specification can be regulated by one or many transcription factors. Here we identify a novel role for one conserved proneural factor, the bHLH protein HLH-3, implicated in the specification of sex-specific ventral cord motor neurons in C. elegans. In the process of characterizing the role of hlh-3 in neural specification, we document that differentiation of the ventral cord type C neurons, VCs, within their motor neuron class, is dynamic in time and space. Expression of VC class-specific and subclass-specific identity genes is distinct through development and dependent on where they are along the A-P axis (and their position in proximity to the vulva). Our characterization of the expression of VC class and VC subclass-specific differentiation markers in the absence of hlh-3 function reveals that VC fate specification, differentiation, and morphology requires hlh-3 function. Finally, we conclude that hlh-3 cell-autonomously specifies VC cell fate.

Cholesterol rich, highly phase separated, naked mole-rat brain lipids are exquisitely sensitive to amyloid induced membrane damage

FRANKEL, D. — 2020-06-05

Naked mole-rats are extraordinarily long-lived rodents that do not develop age-related neurodegenerative diseases. Remarkably, they do not accumulate amyloid plaques, even though their brains contain high concentrations of amyloid beta peptide, even from a young age Therefore, these animals offer an opportunity to investigate mechanisms of resistance against the neurotoxicity of amyloid beta aggregation. Working in this direction, here we examine the composition, phase behaviour, and amyloid beta interactions of naked mole-rat brain lipids. Relative to mouse, naked mole-rat brain lipids are rich in cholesterol and contain sphingomyelin in lower amounts and of shorter chain lengths. Proteins associated with metabolism of ceramides, sphingomyelin and ceramide receptor activity were also found to be decreased in naked mole-rat brain lysates. Correspondingly, we find that naked mole-rat brain lipid membranes exhibit a high degree of phase separation, with the liquid ordered phase occupying up to 80% of the supported lipid bilayer. These observations are consistent with the membrane pacemaker hypothesis of ageing, according to which long-living species have lipid membranes particularly resistant to oxidative damage. However, we found that exposure to amyloid beta disrupts the naked mole-rat brain lipid membranes while those formed from mouse brain lipids exhibit small, well-defined footprints, whereby the amyloid beta penetrates deeply into the lipid membranes. These results suggest that in naked mole-rats the lipid composition of cell membranes may offer neuroprotection through resistance to oxidative processes rather than through mechanical effects.

Mendelian randomization identifies folliculin expression as a mediator of diabetic retinopathy.

Skol, A. D. — 2020-06-11

The goal of the study was to identify genes whose aberrant expression can contribute to diabetic retinopathy. We determined differential gene expression in response to high glucose in lymphoblastoid cell lines derived from matched individuals with type 1 diabetes (T1D) with and without retinopathy. Those genes exhibiting the largest difference in glucose response between individuals with diabetes with and without retinopathy were assessed for association to diabetic retinopathy utilizing genotype data from a genome-wide association study meta-analysis. All genetic variants associated with gene expression (expression Quantitative Trait Loci, eQTLs) of the glucose response genes were tested for association with diabetic retinopathy. We detected an enrichment of the eQTLs from the glucose response genes among small association p-values and identified folliculin (FLCN) as a susceptibility gene for diabetic retinopathy. We show that expression of FLCN in response to glucose was greater in individuals with diabetic retinopathy compared to individuals with diabetes without retinopathy. Three large, independent cohorts of individuals with diabetes revealed an association of FLCN eQTLs to diabetic retinopathy. Mendelian randomization further confirmed a direct positive effect of increased FLCN expression on retinopathy in individuals with diabetes. Together, our studies integrating genetic association and gene expression implicate FLCN as a disease gene for diabetic retinopathy.

Nectar robbing in the trainbearers (Lesbia, Trochilidae)

Igic, B. — 2020-06-12

Many flower visitors engage in floral larceny, a suite of so-called illegitimate visits in which foragers take nectar without providing pollination services. The data on prevalence of illegitimate visits among hummingbirds, as well as the total proportion of foraging and diet that such visits comprise is broadly lacking. Here, we report the occurrence of nectar larceny in both currently recognized species of trainbearers and analyze the proportion of plant visits categorized by mode of interaction as: primary robbing, secondary robbing, theft, and/or pollination. To the best of our knowledge, we provide the first published report identifying robbing in these species. We augment our original field observations using a trove of data from citizen science databases and literature. Although it is difficult to distinguish primary vs. secondary robbing and theft vs. pollination, we conservatively estimate that ca. 40% of the recorded nectar foraging visits involve nectar robbing. Males appear to engage in robbing marginally more than females, but further studies are necessary to confidently examine the multi-way interactions among sex, species, mode of visitation, and other factors. We discuss the significance of these findings in the context of recent developments in study of nectar foraging, larceny, and pollination from both avian and plant perspectives.

Ecological Specialization and Diversification in Birds

Crouch, N. M. A. — 2020-06-13

Ecological specialization is widely thought to influence patterns of species richness by affecting rates at which species multiply and perish. Quantifying specialization is challenging, and using only one or a small number of ecological axes could bias estimates of overall specialization. Here, we calculate an index of specialization, based on seven measured traits, and estimate its effect on speciation and extinction rates in a large clade of birds. We find that speciation rate is independent of specialization, suggesting independence of local ecology and the geographic distributions of populations that promote allopatric species formation. Although some analyses suggest that more specialized species have higher extinction rates, leading to negative net diversification, this relationship is not consistently identified across our analyses. Our results suggest that specialization may drive diversification dynamics only on local scales or in specific clades, but is not generally responsible for macroevolutionary disparity in lineage diversification rates.

Overlapping roles of JIP3 and JIP4 in promoting axonal transport of lysosomes in human iPSC-derived neurons

Gowrishankar, S. — 2020-06-13

The dependence of neurons on microtubule-based motors for the movement of lysosomes over long distances raises questions about adaptations that allow neurons to meet these demands. Recently, JIP3/MAPK8IP3, a neuronally enriched putative adaptor between lysosomes and motors, was identified as a critical regulator of axonal lysosome abundance. In this study, we establish a human induced pluripotent stem cell (iPSC)-derived neuron model for the investigation of axonal lysosome transport and maturation and show that loss of JIP3 results in the accumulation of axonal lysosomes and the Alzheimers disease-related amyloid precursor protein (APP)-derived A{beta}42 peptide. We furthermore reveal an overlapping role of the homologous JIP4 gene in lysosome axonal transport. These results establish a cellular model for investigating the relationship between lysosome axonal transport and amyloidogenic APP processing and more broadly demonstrate the utility of human iPSC-derived neurons for the investigation of neuronal cell biology and pathology.

Biosystematic Studies on the Status of Solanum chilense (Dunal) Reiche

Raduski, A. R. — 2020-06-15

AO_SCPLOWBSTRACTC_SCPLOWO_LIMembers of Solanum sect. Lycopersicum are commonly used as a source of exotic germplasm for improvement of the cultivated tomato, and are increasingly employed in basic research. Although it experienced significant early and ongoing work, the taxonomic status of many wild species in this section has undergone a number of significant revisions, and remains uncertain. C_LIO_LIHere, we examine the taxonomic status of obligately outcrossing Chilean wild tomato (Solanum chilense) using reduced-representation sequencing (RAD-seq), a range of phylogenetic and population genetic analyses, crossing data, and morphological data. C_LIO_LIOverall, each of our analyses provides some weight of evidence that the Pacific coastal populations and Andean inland populations of the currently described S. chilense represent separately evolving populations. C_LIO_LIDespite its vast economic importance, Solanum sect. Lycopersicon still exhibits considerable taxonomic instability. A pattern of under-recognition of outcrossing species may be common across flowering plants. We discuss the possible causes and implications of this observation, with a focus on macroevolutionary inference. C_LI

Novel ACE2-IgG1 fusions with increased activity against SARS-CoV-2

Iwanaga, N. — 2020-06-15

SARS-CoV2, the etiologic agent of COVID-19, uses ACE2 as a cell entry receptor. Soluble ACE2 has been shown to have neutralizing antiviral activity but has a short half-life and no active transport mechanism from the circulation into the alveolar spaces of the lung. To overcome this, we constructed an ACE2-human IgG1 fusion protein with mutations in the catalytic domain of ACE2. This fusion protein contained a LALA mutation that abrogates Fcr{gamma} binding, but retains FcRN binding to prolong the half-life, as well as achieve therapeutic concentrations in the lung lavage. Interestingly, a mutation in the catalytic domain of ACE2, MDR504, completely abrogated catalytic activity, but significantly increased binding to SARS-CoV2 spike protein in vitro. This feature correlated with more potent viral neutralization in a plaque assay. Parental administration of the protein showed stable serum concentrations with a serum half-life of [~] 145 hours with excellent bioavailability in the epithelial lining fluid of the lung. Prophylactic administration of MDR504 significantly attenuated SARS-CoV2 infection in a murine model. These data support that the MDR504 hACE2-Fc is an excellent candidate for pre or post-exposure prophylaxis or treatment of COVID-19.

Lineage-Specific Silencing of PSAT1 Induces Serine Auxotrophy and Sensitivity to Dietary Serine Starvation in Luminal Breast Tumors

Choi, B.-H. — 2020-06-20

SUMMARYA major challenge of targeting metabolism for cancer therapy is pathway redundancy, where multiple sources of critical nutrients can limit the effectiveness of some metabolism-targeted therapies. Here, we analyzed lineage-dependent gene expression in human breast tumors to identify differences in metabolic gene expression that may limit pathway redundancy and create therapeutic vulnerabilities. We found that the serine synthesis pathway gene PSAT1 is the most depleted metabolic gene in luminal breast tumors relative to basal tumors. Low PSAT1 prevents de novo serine biosynthesis and sensitizes luminal breast cancer cells to serine and glycine starvation in vitro and in vivo. This PSAT1 expression disparity—which pre-exists in the putative cells-of-origin of basal and luminal tumors—is due to luminal-specific hypermethylation of the PSAT1 gene. Together, our data demonstrates that luminal breast tumors are auxotrophic for serine and may be uniquely sensitive to dietary serine starvation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Contribution of different host plants to the adult population of western bean cutworm

Bunn, D. C. — 2020-06-20

The western bean cutworm, Striacosta albicosta (Smith) (Lepidoptera: Noctuidae), is historically a pest of both corn (Zea mays L.) and dry beans (Phaseolus sp L.) in the western Great Plains. However, it has recently undergone an eastward range expansion establishing itself across the Corn Belt in twenty-five states and four Canadian provinces. To mitigate the effects of infestation in Michigan, foliar insecticides are used in dry beans whereas management of the pest in corn relies more heavily on the use of Bt-expressing hybrids. In this study stable carbon isotope analysis was used to determine what crop adult moths developed on as larvae with analysis showing that very few of the adult moths developed on dry beans. These results suggest that beans and corn are not suitable as co-refuges and that mainly adults which developed on corn are contributing to the next generation of western bean cutworm in Michigan.

Everyone Can Learn to Meditate: Characterizing a Personalized Connectomic State Space among Meditation Groups

van Doorn, J. — 2020-06-20

Alterations in brain connectivity has been shown for many disease states and groups of people from different levels of cognitive training. To study dynamic functional connectivity, we propose a method for a personalized connectomic state space called Thought Chart. Experienced meditators are an interesting group of healthy subjects for brain connectivity analyses due to their demonstrated differences in resting state dynamics, and altered brain connectivity has been implicated as a potential factor in several psychiatric disorders. Three distinct techniques of meditation are explored: Isha Yoga, Himalayan Yoga, and Vipassana, as well as a meditation-naive group of individuals. All individuals participated in a breath awareness task, an autobiographical thinking task, and one of three different meditation practices according to their expertise, while being recorded by a 64-electrode electroencephalogram (EEG). The functional brain connectivity was estimated using weighted phase lag index (WPLI) and the connectivity dynamics were investigated using a within-individual formulation of Thought Chart, a previously proposed dimensionality reduction method which utilizes manifold learning to map out a state space of functional connectivity. Results showed that the two meditation tasks (breath awareness task and own form of meditation) in all groups were found to have consistently different functional connectivity patterns relative to those of the instructed mind-wandering (IMW) tasks in each individual, as measured using the Hausdorff distance in the state space. The specific meditation state was found to be most similar to the breath awareness state in all groups, as expected in these meditation traditions which all incorporate breath awareness training in their practice trajectory. The difference in connectivity was found to not be solely driven by specific frequency bands. These results demonstrate that the within-individual form of Thought Chart consistently and reliably separates similar tasks among healthy meditators and non-meditators during resting state-like EEG recordings. Unexpectedly, we found the dissimilarity between breath awareness/meditation and IMW, measured via Hausdorff distance, regardless of meditation experience or tradition, with no significant group differences.

Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

Clarke, R. — 2020-06-20

SUMMARYGenetic manipulation of mammalian cells is instrumental to modern biomedical research but is currently limited by poor capabilities of sequentially controlling multiple manipulations in cells. Currently, either highly multiplexed manipulations can be delivered to populations of cells all at one time, or gene regulatory sequences can be engineered to conditionally activate a few manipulations within individual cells. Here, we provide proof-of-principle for a new system enabling multiple genetic manipulations to be executed as a preprogrammed cascade of events. The system leverages the programmability of the S. pyogenes Cas9 RNA-guided nuclease and is based on flexible arrangements of individual modules of activity. The basic module consists of an inactive single guide RNA (sgRNA) - like component that is converted to an active state through the effects of another sgRNA. Modules can be arranged to bring about an algorithmic program of genetic manipulations without the need for engineering cell type specific promoters or gene regulatory sequences. With the expanding diversity of available tools that utilize spCas9 to edit, repress or activate genes, this sgRNA-based system provides multiple levels for interfacing with host cell biology. In addition, ability of the system to progress through multiple modules from episomal plasmid DNA makes it suitable for applications sensitive to the presence of heterologous genomic DNA sequences and broadly applicable to biomedical research and mammalian cell engineering.Competing Interest StatementThe following competing interests are declared for RC, ART, HP, MSM, MR, and BJM as shareholders in Cellgorithmics, Inc., RC, HP, MSM, and BJM as cofounders of Cellgorithmics, Inc., and RC, HP, MSM, and BJM inventors on patent application number PCT/US2018/052211.View Full Text

Fusion Speed of Biomolecular Condensates

Ghosh, A. — 2020-06-23

Biomolecular condensates formed through phase separation have a tendency to fuse. The speed with which fusion occurs is a direct indicator of condensate liquidity, which is key to both cellular functions and diseases. Using a dual-trap optical tweezers setup, we found the fusion speeds of four types of condensates to differ by two orders of magnitude. The order of fusion speed correlates with the fluorescence of Thioflavin T, which in turn reflects the macromolecular packing density inside condensates. Unstructured protein or polymer chains pack loosely and readily rearrange, leading to fast fusion. In contrast, structured protein domains pack more closely and have to break extensive contacts before rearrangement, corresponding to slower fusion. This molecular interpretation for disparate fusion speeds portends a unified understanding of the underlying physicochemical determinants. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=46 SRC="FIGDIR/small/164897v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@e9fb56org.highwire.dtl.DTLVardef@17402f6org.highwire.dtl.DTLVardef@16ea0b8org.highwire.dtl.DTLVardef@1f999ff_HPS_FORMAT_FIGEXP M_FIG The tendency of biomolecular condensates to fuse is key to cellular function and diseases. Using optical tweezers, fluorescence microscopy, and theoretical modeling, Ghosh and Zhou have begun to unravel the molecular origin for disparate fusion speeds among different biomolecular condensates. They found that fusion speed is dictated by macromolecular packing density inside condensates, which can be reported by ThT fluorescence. C_FIG

Central oxytocin signaling inhibits food reward-motivated behaviors and VTA dopamine responses to food-predictive cues

Liu, C. M. — 2020-06-24

Oxytocin potently reduces food intake and is a potential target system for obesity treatment. A better understanding of the behavioral and neurobiological mechanisms mediating oxytocins anorexigenic effects may guide more effective obesity pharmacotherapy development. The present study examined the effects of central (lateral intracerebroventricular [ICV]) administration of oxytocin in rats on motivated responding for palatable food. Various conditioning procedures were employed to measure distinct appetitive behavioral domains, including food seeking in the absence of consumption (conditioned place preference expression), impulsive responding for food (differential reinforcement of low rates of responding), effort-based appetitive decision making (high-effort palatable vs. low-effort bland food), and postingestive reward value encoding (incentive learning). Results reveal that ICV oxytocin potently reduces food-seeking behavior, impulsivity, and effort-based palatable food choice, yet does not influence encoding of postingestive reward value in the incentive learning task. To investigate a potential neurobiological mechanism mediating these behavioral outcomes, we utilized in vivo fiber photometry in ventral tegmental area (VTA) dopamine neurons to examine oxytocins effect on phasic dopamine neuron responses to sucrose-predictive Pavlovian cues. Results reveal that ICV oxytocin significantly reduced food cue-evoked dopamine neuron activity. Collectively, these data reveal that central oxytocin signaling inhibits various obesity-relevant conditioned appetitive behaviors, potentially via reductions in food cue-driven phasic dopamine neural responses in the VTA. HighlightsO_LICentral oxytocin inhibits motivated responding for palatable food reinforcement C_LIO_LICentral oxytocin does not play a role in encoding postingestive reward value C_LIO_LICentral oxytocin blunts VTA dopamine neuron activity in response to food cues C_LI

Increased Expression of Chondroitin Sulfotransferases following AngII may Contribute to Pathophysiology Underlying Covid-19 Respiratory Failure: Impact may be Exacerbated by Decline in Arylsulfatase B Activity

Bhattacharyya, S. — 2020-06-25

The precise mechanisms by which Covid-19 infection leads to hypoxia and respiratory failure have not yet been elucidated. Interactions between sulfated glycosaminoglycans (GAGs) and the SARS-CoV-2 spike glycoprotein have been identified as participating in viral adherence and infectivity. The spike glycoprotein binds to respiratory epithelium through the angiotensin converting enzyme 2 (ACE2) receptor, which endogenously interacts with Angiotensin (Ang) II to yield Angiotensin 1-7. In this report, we show that stimulation of human vascular smooth muscle cells by Ang II leads to increased mRNA expression of two chondroitin sulfotransferases (CHST11 and CHST15), which are required for synthesis of chondroitin 4-sulfate (C4S) and chondroitin 4,6-disulfate (CSE), respectively. Also, increased total sulfated GAGs, increased sulfotransferase activity, and increased expression of the proteoglycans biglycan, syndecan, perlecan, and versican followed treatment by Ang II. Candesartan, an Angiotensin II receptor blocker (Arb), largely, but incompletely, inhibited these increases, and the differences from baseline remained significant. These results suggest that another effect of Ang II also contributes to the increased expression of chondroitin sulfotransferases, total sulfated GAGs, and proteoglycans. We hypothesize that activation of ACE2 may contribute to these increases and suggest that the SARS-CoV-2 spike glycoprotein interaction with ACE2 may also increase chondroitin sulfotransferases, sulfated GAGs, and proteoglycans and thereby contribute to viral adherence to bronchioalveolar cells and to respiratory compromise in SARS-CoV-2 infection.

Inferring cellular trajectories from scRNA-seq using Pseudocell Tracer

Reiman, D. — 2020-06-27

Single cell RNA sequencing (scRNA-seq) can be used to infer a temporal ordering of dynamic cellular states. Current methods for the inference of cellular trajectories rely on unbiased dimensionality reduction techniques. However, such biologically agnostic ordering can prove difficult for modeling complex developmental or differentiation processes. The cellular heterogeneity of dynamic biological compartments can result in sparse sampling of key intermediate cell states. This scenario is especially pronounced in dynamic immune responses of innate and adaptive immune cells. To overcome these limitations, we develop a supervised machine learning framework, called Pseudocell Tracer, which infers trajectories in pseudospace rather than in pseudotime. The method uses a supervised encoder, trained with adjacent biological information, to project scRNA-seq data into a low-dimensional cellular state space. Then a generative adversarial network (GAN) is used to simulate pesudocells at regular intervals along a virtual cell-state axis. We demonstrate the utility of Pseudocell Tracer by modeling B cells undergoing immunoglobulin class switch recombination (CSR) during a prototypic antigen-induced antibody response. Our results reveal an ordering of key transcription factors regulating CSR, including the concomitant induction of Nfkb1 and Stat6 prior to the upregulation of Bach2 expression. Furthermore, the expression dynamics of genes encoding cytokine receptors point to the existence of a regulatory mechanism that reinforces IL-4 signaling to direct CSR to the IgG1 isotype.

Colonization of the murine oropharynx by Streptococcus pyogenes is governed by the Rgg2/3 quorum sensing system

Gogos, A. — 2020-06-30

Streptococcus pyogenes is a human-restricted pathogen most often found in the human nasopharynx. Multiple bacterial factors are known to contribute to persistent colonization of this niche, and many are important in mucosal immunity and vaccine development. In this work, mice were infected intranasally with transcriptional regulator mutants of the Rgg2/3 quorum sensing (QS) system--a peptide-based signaling system conserved in sequenced isolates of S. pyogenes. Deletion of the QS systems transcriptional activator ({Delta}rgg2) dramatically diminished the percentage of colonized mice while deletion of the transcriptional repressor ({Delta}rgg3) increased the percentage of colonized mice compared to wild type. Stimulation of the QS system using synthetic pheromones prior to inoculation did not significantly increase the percentage of animals colonized, indicating that QS-dependent colonization is responsive to the intrinsic conditions within the host upper respiratory tract. Bacterial RNA extracted directly from oropharyngeal swabs and evaluated by quantitative RT-PCR subsequently confirmed QS upregulation within one hour of inoculation. In the nasal-associated lymphoid tissue (NALT), a muted inflammatory response to the {Delta}rgg2 bacteria suggests that their rapid elimination failed to elicit the previously characterized response to intranasal inoculation of GAS. This work identifies a new transcriptional regulatory system governing the ability of S. pyogenes to colonize the nasopharynx and provides knowledge that could help lead to decolonization therapeutics. Author SummaryStreptococcus pyogenes is responsible for a wide spectrum of diseases ranging from common pharyngitis to infrequent invasive infections like necrotizing fasciitis. The ability of this microorganism to persist in the human oropharynx predisposes colonized individuals to a variety of superficial and invasive diseases which lead to significant morbidities and mortality. Identification of the regulatory systems that augment the bacterias ability to colonize the oropharynx provides potential targets against which molecular therapeutics can be designed. Here we show that the Rgg2/3 quorum sensing system, an interbacterial communication system, governs the ability of S. pyogenes to colonize the murine oropharynx. Disruption of the systems transcriptional activator reduced colonization dramatically, eliminated the transcription of two sets of genes known to be activated by the Rgg2/3 system, and tempered the innate immune response seen when S. pyogenes is intranasally infected into the mouse.

Selective Nanotherapeutic Targeting of the Neutrophil Subset Mediating Inflammatory Injury

Bachmaier, K. — 2020-07-02

Inflammatory tissue injury such as acute lung injury (ALI) is a disorder that leads to respiratory failure, a major cause of morbidity and mortality worldwide. Excessive neutrophil influx is a critical pathogenic factor in the development of ALI. Here, we identify the subset of neutrophils that is responsible for ALI and lethality in polymicrobial sepsis. The pro-inflammatory neutrophil subpopulation was characterized by its unique ability to endocytose albumin nanoparticles (ANP), upregulation of pro-inflammatory cytokines and chemokines as well as the excessive production of reactive oxygen species (ROS) in models of endotoxemia and septicemia. ANP delivery of the drug piceatannol, a spleen tyrosine kinase (Syk) inhibitor, to the susceptible subset of neutrophils, prevented ALI and mortality in mice subjected to polymicrobial infection. Targeted inhibition of Syk in ANP-susceptible neutrophils had no detrimental effect on neutrophil-dependent host defense because the subset of ANPlow neutrophils effectively controlled polymicrobial infection. The results show that neutrophil heterogeneity can be leveraged therapeutically to prevent ALI without compromising host defense.Competing Interest StatementABM, AS, KB have interests in the biotechnology company Nano Biotherapeutics Inc.View Full Text

Cigarette Smoke and Nicotine-Containing E-cigarette Vapor Downregulate Lung WWOX Expression Which is Associated with Increased Severity of Murine ARDS

Zeng, Z. — 2020-07-13

RATIONALEA history of chronic cigarette smoking is known to increase risk for ARDS, but the corresponding risks associated with chronic e-cigarette use are largely unknown. The chromosomal fragile site gene, WWOX, is highly susceptible to genotoxic stress from environmental exposures, and thus an interesting candidate gene for the study of exposure-related lung disease. METHODS AND RESULTSLungs harvested from current versus former/never smokers exhibited a 47% decrease in WWOX mRNA levels. Exposure to nicotine-containing e-cigarette vapor resulted in an average 57% decrease in WWOX mRNA levels relative to vehicle treated controls. In separate studies, endothelial (EC)-specific WWOX KO versus wild type mice were examined under ARDS-producing conditions. EC WWOX KO mice exhibited significantly greater levels of vascular leak and histologic lung injury. ECs were isolated from digested lungs of untreated EC WWOX KO mice using sorting by flow cytometry for CD31+CD45- cells. These were grown in culture, confirmed to be WWOX-deficient by RT-PCR and Western blotting, and analyzed by electric cell impedance sensing (ECIS) as well as a FITC dextran transwell assay for their barrier properties during MRSA or LPS exposure. WWOX KO ECs demonstrated significantly greater declines in barrier function relative to wild type cells during either MRSA or LPS treatment as measured by both ECIS and the transwell assay. CONCLUSIONThe increased risk for ARDS observed in chronic smokers may be mechanistically linked, at least in part, to lung WWOX downregulation, and this phenomenon may also manifest in the near future in chronic users of e-cigarettes.

Vitamin D Sufficiency Enhances Differentiation of Patient-Derived Prostate Epithelial Organoids

McCray, T. — 2020-07-17

Vitamin D is an essential steroid hormone that regulates systemic calcium homeostasis and cell fate decisions, including differentiation in many cell types. The prostate gland is hormonally regulated, requiring steroids for proliferation and terminal differentiation of secretory luminal cells. Vitamin D deficiency is associated with higher risk of lethal prostate cancer with an aggressive dedifferentiated pathology, linking vitamin D sufficiency to epithelial differentiation homeostasis. To determine regulation of prostate epithelial differentiation by vitamin D status, patient-derived benign prostate epithelial organoids were maintained in vitamin D deficient (vehicle) or sufficient (10 nM 1,25-dihydroxyvitamin D) conditions and assessed by phenotype and single cell RNA sequencing. Mechanistic validation demonstrated that vitamin D sufficiency promoted organoid growth and accelerated differentiation of lineage-committed cells by inhibiting canonical Wnt activity and Wnt family member DKK3. Wnt dysregulation is a known contributor to aggressive prostate cancer, thus these findings further link vitamin D deficiency to lethal disease.

Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures

Salahudeen, A. A. — 2020-07-27

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5+ cells contained a distinct ITGA6+ITGB4+ mitotic population whose proliferation segregated to a TNFRSF12Ahi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12Ahi subset of FACS-purified ITGA6+ITGB4+ basal cells from human lung or derivative organoids. In vivo, TNFRSF12A+ cells comprised ~10% of KRT5+ basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 "apical-out" organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.

Computational and experimental characterization of the novel ECM glycoprotein SNED1 and prediction of its interactome

Vallet, S. D. — 2020-07-28

The extracellular matrix (ECM) protein SNED1 has been shown to promote breast cancer metastasis and control neural crest cell-specific craniofacial development, but the cellular and molecular mechanisms by which it does so remain unknown. ECM proteins exert their functions by binding to cell surface receptors, sequestering growth factors, and interacting with other ECM proteins, actions that can be predicted using knowledge of proteins sequence, structure and post-translational modifications. Here, we combined in-silico and in-vitro approaches to characterize the physico-chemical properties of SNED1 and infer its putative functions. To do so, we established a mammalian cell system to produce and purify SNED1 and its N-terminal fragment, which contains a NIDO domain. We have determined experimentally SNED1s potential to be glycosylated, phosphorylated, and incorporated into insoluble ECM produced by cells. In addition, we used biophysical and computational methods to determine the secondary and tertiary structures of SNED1 and its N-terminal fragment. The tentative ab-initio model we built of SNED1 suggests that it is an elongated protein presumably able to bind multiple partners. Using computational predictions, we identified 114 proteins as putative SNED1 interactors. Pathway analysis of the newly-predicted SNED1 interactome further revealed that binding partners of SNED1 contribute to signaling through cell surface receptors, such as integrins, and participate in the regulation of ECM organization and developmental processes. Altogether, we provide a wealth of information on an understudied yet important ECM protein with the potential to decipher its functions in physiology and diseases.

Pseudomonas aeruginosa reverse diauxie is an optimized, resource utilization strategy

Carlson, R. — 2020-07-29

Pseudomonas aeruginosa is a globally-distributed bacterium often found in medical infections. The opportunistic pathogen uses a different, carbon catabolite repression (CCR) strategy than many, model microorganisms. It does not utilize a classic diauxie phenotype, nor does it follow common systems biology assumptions including preferential consumption of glucose with an overflow metabolism. Despite these contradictions, P. aeruginosa is competitive in many, disparate environments underscoring knowledge gaps in microbial ecology and systems biology. Physiological, omics, and in silico analyses were used to quantify the P. aeruginosa CCR strategy known as reverse diauxie. An ecological basis of reverse diauxie was identified using a genome-scale, metabolic model interrogated with in vitro omics data. Reverse diauxie preference for lower energy, nonfermentable carbon sources, such as acetate or succinate over glucose, was predicted using a multidimensional strategy which minimized resource investment into central metabolism while completely oxidizing substrates. Application of a common, in silico optimization criterion, which maximizes growth rate, did not predict the reverse diauxie phenotypes. This study quantifies P. aeruginosa metabolic strategies foundational to its wide distribution and virulence.

Discerning the cellular response using statistical discrimination of fluorescence images of membrane receptors

Munaweera, R. — 2020-07-29

We demonstrate a statistical modeling technique to recognize T cell responses to different external environmental conditions using membrane distributions of T cell receptors. We transformed fluorescence images of T cell receptors from each T cell into estimated model parameters of a partial differential equation. The model parameters enabled the construction of an accurate classification model using linear discrimination techniques. We further demonstrated that the technique successfully differentiated immobilized T cells on non-activating and activating surfaces. Compared to machine learning techniques, our statistical technique relies upon robust image-derived statistics and achieves effective classification with a limited sample size and a minimal computational footprint. The technique provides an effective strategy to quantitatively characterize the global distribution of membrane receptors under various physiological and pathological conditions.

Disruption of innate defense responses by endoglycosidase HPSE promotes cell survival

Agelidis, A. — 2020-08-06

The drive to withstand environmental stresses and defend against invasion is a universal trait extant in all forms of life. While numerous canonical signaling cascades have been characterized in detail, it remains unclear how these pathways interface to generate coordinated responses to diverse stimuli. To dissect these connections, we follow heparanase (HPSE), a protein best known for its endoglycosidic activity at the extracellular matrix but recently recognized to drive various forms of late stage disease through unknown mechanisms. Using herpes simplex virus-1 (HSV-1) infection as a model cellular perturbation, we demonstrate that HPSE acts beyond its established enzymatic role to restrict multiple forms of cell-intrinsic defense and facilitate host cell reprogramming by the invading pathogen. We reveal that cells devoid of HPSE are innately resistant to infection and counteract viral takeover through multiple amplified defense mechanisms. With a unique grasp of the fundamental processes of transcriptional regulation and cell death, HPSE represents a potent cellular intersection with broad therapeutic potential.

mRNA induced expression of human angiotensin-converting enzyme 2 in mice for the study of the adaptive immune response to severe acute respiratory syndrome coronavirus 2

Hassert, M. — 2020-08-07

The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic resulting in nearly 20 million infections across the globe, as of August 2020. Critical to the rapid evaluation of vaccines and antivirals is the development of tractable animal models of infection. The use of common laboratory strains of mice to this end is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Interestingly, we did not observe an enhancement of SARS-CoV-2 specific antibody responses with hACE2 induction. Importantly, using this system, we functionally identified the CD4+ and CD8+ peptide epitopes targeted during SARS-CoV-2 infection in H2b restricted mice. Antigen-specific CD8+ T cells in mice of this MHC haplotype primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. The functional identification of these T cell epitopes will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2. The use of this tractable expression system has the potential to be used in other instances of emerging infections in which the rapid development of an animal model is hindered by a lack of host susceptibility factors.

Discovery of a microbially produced small molecule in a host-specific organ

Zink, K. E. — 2020-08-17

The lifelong relationship between the Hawaiian bobtail squid, Euprymna scolopes, and its microbial symbiont, Vibrio fischeri, represents a simplified model system for studying microbiome establishment and maintenance. The bacteria colonize a dedicated symbiotic light organ in the squid, from which bacterial luminescence camouflages the hosts in a process termed counterillumination. The squid hosts hatch without their symbionts, which must be acquired from the ocean amid a diversity of non-beneficial bacteria, so precise molecular communication is required for initiation of the specific relationship. It is therefore likely that there may be specialized metabolites used in the light organ microenvironment to modulate these processes. To identify small molecules that may influence the establishment of this symbiosis, we used imaging mass spectrometry to analyze metabolite production in V. fischeri with altered biofilm production, which correlates directly to colonization capability in its host. Biofilm-Up and Biofilm-Down mutants were compared to a wild-type strain, and masses that were more abundantly produced by the biofilm-up mutant were detected. Using a combination of structure elucidation and synthetic chemistry, one such signal was determined to be a diketopiperazine, cyclo(O_SCPLOWDC_SCPLOW-histidyl-O_SCPLOWLC_SCPLOW-proline). This diketopiperazine modulated luminescence in V. fischeri and, using label-free imaging mass spectrometry, was directly detected in the light organ of the colonized host. This work highlights the continued need for untargeted discovery efforts in host-microbe interactions and showcases the benefits of the squid-Vibrio system for identification and characterization of small molecules that modulate microbiome behaviors. Significance StatementThe complexity of animal microbiomes presents challenges to defining signaling molecules within the microbial consortium and between the microbes and the host. By focusing on the binary symbiosis between Vibrio fischeri and Euprymna scolopes, we have combined genetic analysis with direct imaging to define and study small molecules in the intact symbiosis. We have detected and characterized a diketopiperazine produced by strong biofilm-forming V. fischeri strains that was detectable in the host symbiotic organ, and which influences bacterial luminescence. Biofilm formation and luminescence are critical for initiation and maintenance of the association, respectively, suggesting that the compound may link early and later development stages, providing further evidence that multiple small molecules are important in establishing these beneficial relationships.

Discovery and Development of Novel DNA-PK Inhibitors by Targeting the unique Ku-DNA Interaction

Gavande, N. S. — 2020-08-24

DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) repair pathway and the DNA damage response (DDR). DNA-PK has therefore been pursued for the development of anti-cancer therapeutics in combination with ionizing radiation (IR). We report the discovery of a new class of DNA-PK inhibitors that act via a novel mechanism of action, inhibition of the Ku-DNA interaction. We have developed a series of highly potent and specific Ku-DNA binding inhibitors (Ku-DBis) that block the Ku-DNA interaction and inhibit DNA-PK kinase activity. Ku-DBis directly interact with the Ku and inhibit in vitro NHEJ, cellular NHEJ, and potentiate the activity of IR and radiomimetics. Analysis of Ku-null cells demonstrates that Ku-DBis cellular activity is a direct result of Ku inhibition, as Ku-null cells are insensitive to Ku-DBis. The utility of Ku-DBis was also demonstrated in a CRISPR gene-editing model where we demonstrate that the efficiency of gene insertion events was increased in cells pre-treated with Ku-DBis, consistent with inhibition of NHEJ and activation of homologous recombination to facilitate gene insertion. These data demonstrate the discovery and application of new series of compounds that modulate DNA repair pathways via a unique mechanism of action.

Visualizing the cytosolic delivery of bioconjugated QDs into T cell lymphocytes

Jing, H. — 2020-09-13

The aggregation state and endosomal trapping of engineered nanocarriers once internalized into cells remain poorly characterized. Here, we visualized the membrane penetrating dynamics of semiconductor quantum dots (QDs) into the cytosol of T cells on a single-cell and single-nanoparticle basis. We water solubilized CdSe/CdZnS QDs with polymer encapsulants functionalized with a cell-penetrating peptide composed of an Asp-Ser-Ser (DSS) repeat sequence. T cells tolerated the 24-h incubation with QDs at concentrations of 5 nM or lower. Single-particle imaging demonstrated that the number of internalized nanoparticles was dependent upon the concentration of the probes for both control (peptide-free) and DSS-QDs. DSS-QDs were mostly distributed as monomers, whereas the control QDs were aggregated into clusters. Single-particle tracking using total internal reflection and highly inclined illumination showed that DSS-QDs were stationary near the activating surface and mobile within the cytosol of the T cell. A correlation exhibited between the mobility and aggregation state of individual QD clusters, with monomeric DSS-QDs showing the highest mobility. In addition, monomeric DSS-QDs displayed much faster diffusion than the endosomes. A small-molecule endosome marker confirmed the absence of colocalization between endosomes and DSS-QDs, indicating their endosomal escape. The ability to deliver and track individual QDs in the cytosol of live T cells creates inroads for the optimization of drug delivery and gene therapy through the use of nanoparticles.

COPB2 haploinsufficiency causes a coatopathy with osteoporosis and developmental delay

Marom, R. — 2020-09-15

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures and developmental delay of variable severity. Because the role of COPB2 in bone has not been characterized, we studied the effect of COPB2 deficiency on skeletal development in mice and zebrafish. Copb2+/- mice showed low bone mass and decreased bone strength. In zebrafish, larvae carrying a copb2 heterozygous frameshift variant showed delayed mineralization. copb2-null embryos showed endoplasmic reticulum (ER) and Golgi disorganization, and embryonic lethality. COPB2 siRNA-treated fibroblasts showed delayed collagen trafficking with retention of type I collagen in the ER and Golgi, and altered distribution of Golgi markers. Our data suggest that COPB2 haploinsufficiency leads to disruption of intracellular collagen trafficking and osteoporosis, which may improve with ascorbic acid supplementation. This work highlights the role of COPI complex as a critical regulator of bone mass and identifies a new form of coatopathy due to COPB2 deficiency.

The healing potential of wild type and recombinant S100A8 to attenuate skin wound inflammation

Akinshipo, A. W. O. — 2020-09-17

BackgroundWounds represent a major health burden in our society and poorly healing wounds are a significant clinical problem worldwide, During the acute inflammatory, neutrophils which are normal wound scavengers seems to create additional tissue destruction and promote scar formation. This project examined the utility of using pluronic gel to deliver ala42S100A8, a peptide that repels neutrophils, to wounds, allowing more regenerative repair. MethodExcisional wound models on female BALB/c mice were made and 4 treatments including pluronic gel only group, Wild type S100A8 (1, 2, and 4g) with Pluronic gel, and ala42S100A8 (1, 2, and 4g) with Pluronic gel were applied to the wounds. Wounds were harvested at day 1 and day 3. Myeloperoxidase (MPO) protein level was examined using an ELISA kit and cytokine protein expression of CXCL1 (GRO-1), CXCL2 (MIP-2). IL-6, and TNF- was determined using a multiplex ELISA kit. ResultsMPO level in Pluronic gel treated wounds at day 1 was significantly higher than that in control, suggesting that the Pluronic gel itself causes increased inflammation in wounds, while treatment with 1g of s100A8 or 1 and 4g of ala42S100A8 seemed to decrease MPO at day 1 compared to the Pluronic gel treated wounds. Treatment with 1g of s100A8 also led to a decrease IL-6 and TNF- production at day 1 when compared to the Pluronic gel group, although no statistical difference was observed ConclusionsOur findings strongly suggest that wound inflammation is reduced by treatment with 1ug of S100A8. As such, this study provides proof-of-principal for further investigations of S100A8/ala42S100A8 as a wound therapeutic. Additional studies with lower doses and increased sample size, along with the use of alternative delivery systems, will provide important information about the utility of this approach.

Extreme Fuzzy Association of an Intrinsically Disordered Protein with Acidic Membranes

Hicks, A. — 2020-09-17

Many physiological and pathophysiological processes, including Mycobacterium tuberculosis (Mtb) cell division, may involve fuzzy membrane association by proteins via intrinsically disordered regions. The fuzziness is extreme when the conformation and pose of the bound protein and the composition of the proximal lipids are all highly dynamic. Here we tackled the challenge in characterizing the extreme fuzzy membrane association of the disordered, cytoplasmic N-terminal region (NT) of ChiZ, an Mtb divisome protein, by combining solution and solid-state NMR spectroscopy and molecular dynamics simulations. In a typical pose, NT is anchored to acidic membranes by Arg residues in the midsection. Competition for Arg interactions between lipids and acidic residues, all in the first half of NT, makes the second half more prominent in membrane association. This asymmetry is accentuated by membrane tethering of the downstream transmembrane helix. These insights into sequence-interaction relations may serve as a paradigm for understanding fuzzy membrane association.

Single-molecule interaction microscopy reveals antibody binding kinetics

Perera, T. — 2020-09-22

Single-molecule imaging has provided new insights on weak transient biomolecular interactions with micromolar to millimolar affinity. However, the limited duration of observation has hindered the study of strong and reversible interactions with sub-nanomolar affinity. We report single-molecule interaction microscopy (SMIM), which combines point accumulation for imaging in nanoscale topography (PAINT) with extended imaging durations that enables the study of antibody binding kinetics in the cellular environment. SMIM revealed heterogeneous binding kinetics and the effect of concentration and antibody valency on the association and dissociation rates on antibody-antigen interactions in their cellular environments. We thereby demonstrate SMIM as a versatile single-molecule technique for studying strong, transient biomolecular interactions.

Tethering-facilitated DNA 'opening' and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein

Paul, D. — 2020-09-29

XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively opening these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a kinetic gating mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how opening is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed open undamaged DNA in solution and that such opening can largely occur without one or the other of the {beta}-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound open DNA adopts multiple conformations in solution notwithstanding the DNAs original structure or the {beta}-hairpins. Molecular dynamics simulations reveal compensatory roles of the {beta}-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA opening of undamaged sites and the dynamic nature of open DNA may shed light on how the protein functions within and beyond NER in cells.

Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein

Tavakoli, A. — 2020-09-29

Biomolecular structural changes upon binding/unbinding are key to their functions. However, characterization of such dynamical processes with high spatial and temporal resolutions is difficult as it requires ways to rapidly trigger the assembly/disassembly as well as ways to monitor the structural changes over time. Recently, various chemical strategies have been developed to use light to trigger changes in oligonucleotide structures, thereby their activities. Here we report that photoswitchable DNA can be used to modulate the DNA binding of the Rad4/XPC DNA repair complex using light. Rad4/XPC specifically binds to diverse helix-destabilizing/distorting lesions including bulky organic adducts and functions as a key initiator for the eukaryotic nucleotide excision repair (NER) pathway. We show that the 6-nitropiperonyloxymethyl (NPOM)-modified DNA is recognized by the Rad4 protein as a specific substrate and that the specific binding can be abolished by light-induced cleavage of the NPOM group from DNA in a dose-dependent manner. Fluorescence lifetime-based analyses of the DNA conformations suggest that free NPOM-DNA retains B-DNA-like conformations despite its bulky NPOM adduct, but Rad4-binding renders it to be heterogeneously distorted. Subsequent extensive conformational searches and molecular dynamics simulations demonstrate that NPOM in DNA can be housed in the major groove of the DNA, with stacking interactions among the nucleotide pairs remaining largely unperturbed and thus retaining overall B-DNA conformation. Our work suggests that photoactivable DNA can be used as a DNA lesion surrogate to study DNA repair mechanisms such as nucleotide excision repair. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/313114v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f31fc7org.highwire.dtl.DTLVardef@ab512dorg.highwire.dtl.DTLVardef@15e8493org.highwire.dtl.DTLVardef@51f58d_HPS_FORMAT_FIGEXP M_FIG Graphical Summary This work shows that a photolabile 6-nitropiperonyloxymethyl (NPOM)-modified DNA is specifically recognized by the Rad4/XPC damage sensor protein complex that initiates the nucleotide excision repair pathway; light-induced cleavage of NPOM abolishes the specific binding to Rad4/XPC. C_FIG

Epigenetic clock and DNA methylation analysis of porcine models of aging and obesity

Schachtschneider, K. M. — 2020-10-01

DNA-methylation profiles have been used successfully to develop highly accurate biomarkers of age, epigenetic clocks, for many species. Using a custom methylation array, we generated DNA methylation data from n=238 porcine tissues including blood, bladder, frontal cortex, kidney, liver and lung, from domestic pigs (Sus scrofa domesticus) and minipigs (Wisconsin Miniature Swine). We present 4 epigenetic clocks for pigs that are distinguished by their compatibility with tissue type (pan-tissue and blood clock) and species (pig and human). Two dual-species human-pig pan-tissue clocks accurately measure chronological age and relative age, respectively. We also characterized CpGs that differ between minipigs and domestic pigs. Strikingly, several genes implicated by our epigenetic studies of minipig status overlap with genes (ADCY3, TFAP2B, SKOR1, and GPR61) implicated by genetic studies of body mass index in humans. In addition, CpGs with different levels of methylation between the two pig breeds were identified proximal to genes involved in blood LDL levels and cholesterol synthesis, of particular interest given the minipigs increased susceptibility to cardiovascular disease compared to domestic pigs. Thus, inbred differences of domestic and minipigs may potentially help to identify biological mechanisms underlying weight gain and aging-associated diseases. Our porcine clocks are expected to be useful for elucidating the role of epigenetics in aging and obesity, and the testing of anti-aging interventions.

mTORC2 combats cellular stress and potentiates immunity during viral infection

Suryawanshi, R. K. — 2020-10-10

Herpes simplex virus type 1 (HSV-1) causes ocular and orofacial infections, which are generally well controlled by the host and nonlethal. In rare cases, HSV-1 causes encephalitis, which leads to permanent brain injuries, memory loss or even death. Host factors protect the organism from viral infections by activating the immune response. However, the factors that confer neuroprotection during viral encephalitis are unknown. Here we show that mammalian target of rapamycin complex 2 (mTORC2) is essential for the host survival of ocular HSV-1 infections in vivo. We found that the loss of mTORC2 causes systemic HSV-1 infection not only because of weak innate and adaptive immune responses but also due to increased ocular and neuronal cell death, which becomes lethal over time. Furthermore, we found that mTORC2 mediates cell survival channels through the inactivation of the proapoptotic factor FoxO3a. Our results demonstrate how mTORC2 potentiates host defenses against viral infections as well as implicating mTORC2 as a necessary host factor for survival. We anticipate our findings may help develop new therapeutic window for severe HSV-1 infections, such as herpes simplex encephalitis.

Characterization of Constitutive macro-ER-phagy

Lipatova, Z. — 2020-10-21

Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health. Author SummaryAccumulation of excess proteins can lead to their aggregation, which in turn can cause multiple disorders, notably neurodegenerative disease. Nutritional and endoplasmic-reticulum (ER) stress stimulate autophagy and ER-associated degradation (ERAD) to clear excess and misfolded proteins, respectively. However, not much is known about clearance of excess proteins during normal growth. We have previously shown that excess integral-membrane proteins are cleared from the ER by macro-autophagy during normal growth of yeast cells. Here we characterize this pathway as constitutive ER-phagy. While this pathway shares machinery of core Atgs and autophagosomes with nutritional stress-induced ER-phagy, it differs from the latter: It is independent of the stress response and of receptors needed for stress-induced ER-phagy, and while stress-induced ER-phagy is not discriminatory, constitutive ER-phagy has specific cargos. However, when constitutive ER-phagy fails, machinery specific to stress-induced ER-phagy can process its cargo. Moreover, constitutive ER-phagy is also not dependent on ER-stress or the unfolded protein response (UPR) stimulated by this stress, although its failure elicits UPR. Finally, constitutive ER-phagy and ERAD can partially process each others cargo upon failure. In summary, constitutive ER-phagy, which clears excess integral-membrane proteins from the ER during normal growth does not require nutritional or ER stress for its function.

Glycolysis Inhibition Regulates Endothelial Junctions by Perturbing Actin and Focal Adhesions

Leckband, D. — 2020-10-22

One of the central functions of the endothelium is to maintain a vascular barrier that prevents fluid leakiness and immune cell influx from the circulating blood into the tissue. The barrier integrity of the endothelium is largely controlled by adherens junctions (AJs) and the key AJ molecule VE-cadherin, which maintains cell cohesion via homotypic trans-interaction with VE-cadherin molecules on neighboring endothelial cells. Tension is required to maintain junction homeostasis, but little is known about the role of endothelial metabolism and bioenergetics in regulating junctional forces. Because glycolysis is the main source of ATP generation in endothelial cells, we examined the bioenergetic control of the mechanics of VE-cadherin junctions, by focusing on the glycolysis regulatory enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Results from traction force imbalance measurements and a VE-cadherin tension sensor revealed that inhibiting PFKFB3 significantly reduced the average junctional tension and the force on VE-cadherin complexes. The decrease in tension was largely due to mechanical changes distal from the cell-cell contacts. Specifically, inhibiting glycolysis perturbs focal adhesions and disrupts actin organization, directly impacting the net force on intercellular contacts. These findings identify a critical role of cellular metabolism for the mechanics and integrity of vascular endothelial junctions, by maintaining global cell mechanics. Statement of SignificanceThis study examines how forces at intercellular junctions are bioenergetically regulated. Results reveal altered mechanical force generation and transmission due to the breakdown of stress-transmitting fibers during lung injury. These junctions control the barrier function of the vascular endothelium, which requires tight inter cellular adhesions to prevent fluid and macromolecules from passing through the endothelial barrier. We determined how the availability of ATP affects the tension between human endothelial cells, by regulating forces produced remotely from the junctions. These global changes alter both the force at the junctions themselves, and the force transmitted across the entire cell through actin fibers.

Striking parallels between dorsoventral patterning in Drosophila and Gryllus reveal a complex evolutionary history behind a model gene regulatory network

Pechmann, M. — 2020-10-28

Dorsoventral pattering relies on Toll and BMP signalling in all insects studied so far, with variations in the relative contributions of both pathways. Drosophila and the beetle Tribolium share extensive dependence on Toll, while representatives of more distantly related lineages like the wasp Nasonia and bug Oncopeltus rely more strongly on BMP signalling. Here, we show that in the cricket Gryllus bimaculatus, an evolutionarily distant outgroup, Toll has, like in Drosophila, a direct patterning role for the ventral half of the embryo. In addition Toll polarizes BMP signalling, although this does not involve the conserved BMP inhibitor Sog/Chordin. Finally, Toll activation relies on ovarian patterning mechanisms with striking similarity to Drosophila. Our data suggest two surprising hypotheses: 1) that Tolls patterning function in Gryllus and Drosophila is the result of convergent evolution or 2) a Drosophila-like system arose early in insect evolution, and was extensively altered in multiple independent lineages.

Rapid High Throughput Whole Genome Sequencing of SARS-CoV-2 by using One-step RT-PCR Amplification with Integrated Microfluidic System and Next-Gen Sequencing

Hang, J. — 2020-11-05

The long-lasting global COVID-19 pandemic demands timely genomic investigation of SARS-CoV-2 viruses. Here we report a simple and efficient workflow for whole genome sequencing utilizing one-step RT-PCR amplification on a microfluidic platform, followed by MiSeq amplicon sequencing. The method uses Fluidigm IFC and instruments to amplify 48 samples with 39 pairs of primers in a single step. Application of this method on RNA samples from both viral isolate and clinical specimens demonstrate robustness and efficiency of this method in obtaining the full genome sequence of SARS-CoV-2.

Visualising Cholesterol in Brain by On-Tissue Derivatisation and Quantitative Mass Spectrometry Imaging

Angelini, R. — 2020-11-07

Despite being a critical molecule for neurobiology and brain health, mass spectrometry imaging (MSI) of cholesterol has been under reported compared to other lipids, due to the difficulty in ionising the sterol molecule. In the present work we have employed an on-tissue enzyme-assisted derivatisation strategy to improve detection of cholesterol in brain tissue sections. We report distribution and levels of cholesterol across specific brain structures of the mouse brain, in a model of Niemann-Pick type C1 (NPC1) disease, and during brain development. MSI revealed how cholesterol changes during development and that in the adult is highest in pons and medulla of the brain stem. Cholesterol was significantly reduced in the corpus callosum and other brain regions in the Npc1 null mouse, confirming hypomyelination at the molecular level. Our study demonstrates the potential of MSI to the study of sterols in neuroscience.

Synaptic integration by co-localized G protein-coupled receptors in presynaptic terminals

Church, E. — 2020-11-12

In presynaptic terminals, membrane-delimited Gi/o-mediated presynaptic inhibition is ubiquitous and acts through G{beta}{gamma} to inhibit Ca2+ entry, or directly at SNARE complexes to inhibit Ca2+-dependent synaptotagmin-SNARE complex interactions. At CA1-subicular presynaptic terminals 5-HT1B and GABAB receptors colocalize. GABAB receptors inhibit Ca2+ entry, whereas 5-HT1B receptors target SNARE complexes. We demonstrate in male and female rats that GABAB receptors receptors alter Pr, whereas 5-HT1B receptors reduce evoked cleft glutamate concentrations allowing differential inhibition of AMPA and NMDA receptor EPSCs. This reduction in cleft glutamate concentration was confirmed by imaging glutamate release using a genetic sensor (iGluSnFR).Simulations of glutamate release and postsynaptic glutamate receptor currents were made. We tested effects of changes in vesicle numbers undergoing fusion at single synapses, relative placement of fusing vesicles and postsynaptic receptors, and the rate of release of glutamate from a fusion pore. Experimental effects of Pr changes, consistent with GABAB receptor effects, were straightforwardly represented by changes in numbers of synapses. The effects of 5-HT1B receptor-mediated inhibition are well-fit by simulated modulation of the release rate of glutamate into the cleft. Colocalization of different actions of GPCRs provide synaptic integration within presynaptic terminals. Train-dependent presynaptic Ca2+ accumulation forces frequency-dependent recovery of neurotransmission during 5-HT1B receptor activation. This is consistent with competition between Ca2+-synaptotagmin and G{beta}{gamma} at SNARE complexes. Thus, stimulus trains in 5-HT1B receptor agonist unveil dynamic synaptic modulation and a sophisticated hippocampal output filter that itself is modulated by colocalized GABAB receptors which alter presynaptic Ca2+. In combination these pathways allow complex presynaptic integration. Significance StatementTwo G protein coupled receptors colocalize at presynaptic sites, to mediate presynaptic modulation by G{beta}{gamma}, but one - a GABAB receptor inhibits Ca2+ entry while another - a 5-HT1B receptor competes with Ca2+-synaptotagmin binding to the synaptic vesicle machinery. We have investigated downstream effects of signaling and integrative properties of these receptors. Their effects are profoundly different. GABAB receptors alter Pr leaving synaptic properties unchanged, while 5-HT1B receptors fundamentally change properties of synaptic transmission, modifying AMPA receptor but sparing NMDA receptor responses. Co-activation of these receptors allows synaptic integration because of convergence of GABAB receptor alteration on Ca2+ and the effect of this altered Ca2+ signal on 5-HT1B receptor signaling. This presynaptic convergence provides a novel form of synaptic integration.

96-Well Oxygen Control Using a 3D-Printed Device

Szmelter, A. — 2020-11-12

Oxygen concentration varies tremendously within the body and has proven to be a critical variable in cell differentiation, proliferation, and drug metabolism among many other physiological processes. Currently, researchers study the gass role in biology using low-throughput gas-control incubators or hypoxia chambers in which all cells in a vessel are exposed to a single oxygen concentration. Here, we introduce a device which can simultaneously deliver 12 unique oxygen concentrations to cells in a 96-well plate and seamlessly integrate into biomedical research workflows. The device inserts into 96-well plates and delivers gas to the headspace thus avoiding undesirable contact with media. This simple approach isolates each well using gas-tight pressure resistant gaskets effectively creating 96 "mini-incubators". Each of the twelve columns of the plate is supplied by a distinct oxygen concentration from a gas-mixing gradient generator supplied by two feed gases. The wells within each column are then supplied by an equal flow-splitting distribution network. Using equal feed flow rates, concentrations ranging from 0.6% to 20.5% were generated within a single plate. A549 lung carcinoma cells were then used to show that O2 levels below 9% caused a stepwise increase in cell death for cells treated with the hypoxia-activated anti-cancer drug Tirapirizamine (TPZ). Additionally, the 96-well plate was further leveraged to simultaneously test multiple TPZ concentrations over an oxygen gradient and generate a 3D dose response landscape. The results presented here show how microfluidic technologies can be integrated into, rather than replace, ubiquitous biomedical labware allowing for increased throughput oxygen studies.

Sex-specific plasticity and the nutritional geometry of insulin-signaling gene expression in Drosophila melanogaster

McDonald, J. M. C. — 2020-11-16

1.BackgroundSexual-size dimorphism (SSD) is replete among animals, but while the selective pressures that drive the evolution of SSD have been well studied, the developmental mechanisms upon which these pressures act are poorly understood. Ours and others research has shown that SSD in Drosophila reflects elevated levels of nutritional plasticity in females versus males, such that SSD increases with dietary intake and body size, a phenomenon called sex-specific plasticity (SSP). Additional data indicate that while body size in both sexes responds to variation in protein level, only female body size is sensitive to variation in carbohydrate level. Here we explore whether these difference in sensitivity at the morphological level are reflected by differences in how the insulin/IGF-signaling (IIS) and TOR-signaling pathways respond to changes in carbohydrates and proteins in females versus males, using a nutritional geometry approach. ResultsThe IIS-regulated transcripts of 4E-BP and InR most strongly correlated with body size in females and males respectively, but neither responded to carbohydrate level and so could not explain the sex-specific response to body size to dietary carbohydrate. Transcripts regulated by TOR-signaling did, however, respond to dietary carbohydrate in a sex-specific manner. In females, expression of dILP5 positively correlated with body size, while expression of dILP2,3 and 8, was elevated on diets with a low concentration of both carbohydrate and protein. In contrast, we detected lower levels of dILP2 and 5 protein in the brains of females fed on low concentration diets. We could not detect any effect of diet on dILP expression in males. ConclusionAlthough females and males show sex-specific transcriptional responses to changes in protein and carbohydrate, the patterns of expression do not support a simple model of the regulation of body-size SSP by either insulin-or TOR-signaling. The data also indicate a complex relationship between carbohydrate and protein level, dILP expression and dILP peptide levels in the brain. In general, diet quality and sex both affect the transcriptional response to changes in diet quantity, and so should be considered in future studies that explore the effect of nutrition on body size.

A repurposed, blood gene signature is associated with poor outcomes in SARS-CoV-2

Juan-Guardela, B. M. — 2020-11-23

Poor outcomes after SARS-CoV-2 infection are difficult to predict. Survivors may develop pulmonary fibrosis. We previously identified a 52-gene signature in peripheral blood, predictive of mortality in Idiopathic Pulmonary Fibrosis. In this study, we analyzed this signature in SARS-CoV-2 infected individuals and identified genomic risk profiles with significant differences in outcomes. Analysis of single cell expression data shows that monocytes, red blood cells, neutrophils and dendritic cells are the cellular source of the high risk gene signature.

Effect size, sample size and power of forced swim test assays in mice: Guidelines for investigators to optimize reproducibility

Smalheiser, N. R. — 2020-11-26

A recent flood of publications has documented serious problems in scientific reproducibility, power, and reporting of biomedical articles, yet scientists persist in their usual practices. Why? We examined a popular and important preclinical assay, the Forced Swim Test (FST) in mice used to test putative antidepressants. Whether the mice were assayed in a naive state vs. in a model of depression or stress, and whether the mice were given test agents vs. known antidepressants regarded as positive controls, the mean effect sizes seen in the experiments were indeed extremely large (1.5 - 2.5 in Cohens d units); most of the experiments utilized 7-10 animals per group which did have adequate power to reliably detect effects of this magnitude. We propose that this may at least partially explain why investigators using the FST do not perceive intuitively that their experimental designs fall short -- even though proper prospective design would require ~21-26 animals per group to detect, at a minimum, large effects (0.8 in Cohens d units) when the true effect of a test agent is unknown. Our data provide explicit parameters and guidance for investigators seeking to carry out prospective power estimation for the FST. More generally, altering the real-life behavior of scientists in planning their experiments may require developing educational tools that allow them to actively visualize the inter-relationships among effect size, sample size, statistical power, and replicability in a direct and intuitive manner.

Viscoelasticity of biomolecular condensates conforms to the Jeffreys model

Zhou, H.-X. — 2020-11-27

Biomolecular condensates, largely by virtue of their material properties, are revolutionizing biology, and yet physical understanding of these properties is lagging. Here I show that the viscoelasticity of condensates can be captured by a simple model, comprising a component where shear relaxation is an exponential function of time and a component that is purely viscous (corresponding to instantaneous shear relaxation). Modulation of intermolecular interactions, e.g., by adding salt, can disparately affect the two components, such that the exponentially-relaxing component may dominate at low salt whereas the purely viscous component may dominate at high salt. Condensates have a tendency to fuse, with the dynamics accelerated by surface tension and impeded by viscosity. For fast-fusion condensates, shear relaxation may become rate-limiting. These insights help narrow the gap in understanding between the biology and physics of biomolecular condensates.

V-NeuroStack: 3D Time Stacks for Identifying Patterns in Calcium Imaging Data

Naik, A. — 2020-12-04

BackgroundUnderstanding functional correlations between the activities of neuron populations is vital for the analysis of neuronal networks. Analyzing large-scale neuroimaging data obtained from hundreds of neurons simultaneously poses significant visualization challenges. We developed V-NeuroStack, a novel network visualization tool to visualize data obtained using calcium imaging of spontaneous activity of cortical neurons in a mouse brain slice. New MethodV-NeuroStack creates 3D time stacks by stacking 2D time frames for a period of 600 seconds. It provides a web interface that enables exploration and analysis of data using a combination of 3D and 2D visualization techniques. Comparison with existing MethodsPrevious attempts to analyze such data have been limited by the tools available to visualize large numbers of correlated activity traces. V-NeuroStack can scale data sets with at least a few thousand temporal snapshots. ResultsV-NeuroStacks 3D view is used to explore patterns in the dynamic large-scale correlations between neurons over time. The 2D view is used to examine any timestep of interest in greater detail. Furthermore, a dual-line graph provides the ability to explore the raw and first-derivative values of a single neuron or a functional cluster of neurons. ConclusionsV-NeuroStack enables easy exploration and analysis of large spatio-temporal datasets using two visualization paradigms: (a) Space-Time cube (b)Two-dimensional networks, via web interface. It will support future advancements in in vitro and in vivo data capturing techniques and can bring forth novel hypotheses by permitting unambiguous visualization of large-scale patterns in the neuronal activity data.

A Streptococcus quorum sensing system enables suppression of innate immunity

Rahbari, K. M. — 2020-12-04

Some bacterial pathogens utilize cell-cell communication systems, such as quorum sensing (QS), to coordinate genetic programs during host colonization and infection. The human-restricted pathosymbiont Streptococcus pyogenes (Group A Streptococcus, GAS) uses the Rgg2/Rgg3 QS system to modify the bacterial surface, enabling biofilm formation and lysozyme resistance. Here, we demonstrate that innate immune cell responses to GAS are substantially altered by the QS status of the bacteria. We found that macrophage activation, stimulated by multiple agonists and assessed by cytokine production and NF{kappa}B activity, was substantially suppressed upon interaction with QS-active GAS but not QS-inactive bacteria. Neither macrophage viability nor bacterial adherence were seen as different between QS activity states, yet TNF, IL-6, and IFN{beta} levels and NF{kappa}B reporter activity were drastically lower following infection with QS-active GAS. Suppression required contact between viable bacteria and macrophages. A QS-regulated biosynthetic gene cluster (BGC) in the GAS genome, encoding several putative enzymes, was also required for macrophage modulation. Our findings suggest a model wherein upon contact with macrophages, QS-active GAS produce a BGC-derived factor capable of suppressing inflammatory responses. The suppressive capability of QS-active GAS is abolished after treatment with a specific QS inhibitor. These observations suggest that interfering with the ability of bacteria to collaborate via QS can serve as a strategy to counteract microbial efforts to manipulate host defenses. ImportanceStreptococcus pyogenes is restricted to human hosts and commonly causes superficial diseases such as pharyngitis; it can also cause severe and deadly manifestations including necrotizing skin disease or severe post-infectious sequelae like rheumatic heart disease. Understanding the complex mechanisms used by this pathogen to manipulate host defenses could aid in developing new therapeutics to treat infections. Here, we examine the impact of a bacterial cell-cell communication system, which is highly conserved across S. pyogenes, on host innate immune responses. We find that S. pyogenes uses this system to suppress macrophage pro-inflammatory cytokine responses. Interference with this communication system could serve as a strategy to disarm bacteria and maintain an effective immune response.

Regions of Highly Recurrent Electrogram Morphology at Sites of Low Cycle Length Accurately Reflect Arrhythmogenic Substrate for Atrial Fibrillation - Implications For a New, Mechanism Guided Therapeutic Approach for Atrial Fibrillation

Yoo, S. — 2020-12-11

BackgroundAlthough atrial electrograms (EGMs) are thought to reflect pathophysiological substrate for atrial fibrillation (AF), it is not known which electrograms are suitable targets during AF ablation. We hypothesized that electrogram morphology recurrence (EMR) better reflects arrhythmogenic AF substrate than traditional frequency and complexity measures of AF. In a canine rapid atrial pacing (RAP) model of AF, we assessed the relationship between EMR and traditional AF electrogram measures, rotational activity in the atria, fibrosis, myofiber orientation and parasympathetic innervation. MethodsPersistent AF was induced in 13 dogs by RAP for 6-8 weeks. High-density epicardial mapping (117 electrodes) was performed in six atrial sub-regions. EMR measures Recurrence percentage (Rec%) and cycle length of the most frequent electrogram morphology (CLR), Fractionated Interval (FI), Organization Index (OI), Dominant Frequency (DF) and Shannons Entropy (ShEn) were analyzed before and after atropine administration. Myocyte fiber orientation, amount of fibrosis and spatial distribution of parasympathetic nerve fibers were quantified. ResultsRec% was greatest in the appendages, and CLR was lowest in the posterior left atrium. Rec%/CLR correlated with FI, OI and the complexity measure ShEn, but not with DF. All electrogram measures were poorly correlated with fibrosis and myofiber anisotropy. Rec% correlated closely with stability of rotational activity. Unlike other measures, Rec% correlated closely with spatial heterogeneity of parasympathetic nerve fibers; this was reflected in CLR response to atropine. ConclusionEMR correlates closely with stability of rotational activity and with the pattern of atrial parasympathetic innervation. CLR may therefore be a viable therapeutic target in persistent AF.

Rapid SARS-CoV-2 Detection and Classification Using Phase Imaging with Computational Specificity

Goswami, N. — 2020-12-15

Efforts to mitigate the COVID-19 crisis revealed that fast, accurate, and scalable testing is crucial for curbing the current impact and that of future pandemics. We propose an optical method for directly imaging unlabeled viral particles and using deep learning for detection and classification. An ultrasensitive interferometric method was used to image four virus types with nanoscale optical pathlength sensitivity. Pairing these data with fluorescence images for ground truth, we trained semantic segmentation models based on U-Net, a particular type of convolutional neural network. The trained network was applied to classify the viruses from the interferometric images only, containing simultaneously SARS-CoV-2, H1N1 (influenza-A), HAdV (adenovirus), and ZIKV (Zika). Remarkably, due to the nanoscale sensitivity in the input data, the neural network was able to identify SARS-CoV-2 vs. the other viruses with 96% accuracy. The inference time for each image is 60 ms, on a common graphic processing unit. This approach of directly imaging unlabeled viral particles may provide an extremely fast test, of less than a minute per patient. As the imaging instrument operates on regular glass slides, we envision this method as potentially testing on patient breath condensates. The necessary high throughput can be achieved by translating concepts from digital pathology, where a microscope can scan hundreds of slides automatically. One Sentence SummaryThis work proposes a rapid (<1 min.), label-free testing method for SARS-CoV-2 detection, using quantitative phase imaging and deep learning.

MiMeNet: Exploring Microbiome-Metabolome Relationships using Neural Networks

Reiman, D. — 2020-12-16

The advance in microbiome and metabolome studies has generated rich omics data revealing the involvement of the microbial community in host disease pathogenesis through interactions with their host at a metabolic level. However, the computational tools to uncover these relationships are just emerging. Here, we present MiMeNet, a neural network framework for modeling microbe-metabolite relationships. Using ten iterations of 10-fold cross-validation on three paired microbiome-metabolome datasets, we show that MiMeNet more accurately predicts metabolite abundances (mean Spearman correlation coefficients increase from 0.108 to 0.309, 0.276 to 0.457, and -0.272 to 0.264) and identifies more well-predicted metabolites (increase in the number of well-predicted metabolites from 198 to 366, 104 to 143, and 4 to 29) compared to state-of-art linear models for individual metabolite predictions. Additionally, we demonstrate that MiMeNet can group microbes and metabolites with similar interaction patterns and functions to illuminate the underlying structure of the microbe-metabolite interaction network, which could potentially shed light on uncharacterized metabolites through "Guilt by Association". Our results demonstrated that MiMeNet is a powerful tool to provide insights into the causes of metabolic dysregulation in disease, facilitating future hypothesis generation at the interface of the microbiome and metabolomics.

A novel family of nonribosomal peptides modulate collective behavior in Pseudovibrio bacteria isolated from marine sponges

Ioca, L. P. — 2020-12-15

Collective behavior is a common feature of life. Although swarming motility and biofilms are opposed collective behaviors, both contribute to bacterial survival and host colonization. We have identified a link between motility/biofilms and a nonribosomal peptide synthetase-polyketide synthase gene cluster family (ppp) conserved in Pseudovibrio and Pseudomonas Proteobacteria known to interact with diverse eukaryotes. After developing reverse genetics for Pseudovibrio, we discovered two pseudovibriamide families, heptapeptides with a reversal in chain polarity via an ureido linkage 1-6 and related nonadepsipeptides 7-12. Imaging mass spectrometry showed that 1 was excreted whereas 7 was colony-associated. Deletion of pppA abolished production of 1-12 leading to reduced motility and increased biofilm production. pppD mutants that produced only 1-6 showed motility comparable to the wild-type and reduced biofilm formation, indicating that the excreted heptapeptides play a role in promoting motility. In contrast to lipopeptides widely known to affect swarming and biofilms, pseudovibriamides are not surfactants. Our results expand current knowledge on metabolites mediating bacterial collective behavior. Moreover, the establishment of reverse genetics will enable future exploration of the ecological and biotechnological potential of Pseudovibrio bacteria which have been proposed to contribute to marine sponge health. SignificanceBacteria contribute to health and disease of plants and animals. Specialized metabolites produced by bacteria are important in mediating their behavior and the colonization of their hosts. We have identified a conserved gene cluster family in Pseudovibrio and Pseudomonas bacteria known to colonize marine animals and terrestrial plants, respectively. Using Pseudovibrio as a model, we show the encoded metabolites, which we termed pseudovibriamides, promote motility and decrease biofilms. In contrast to lipopeptides widely known to affect motility/biofilms, pseudovibriamides are not surfactants, but instead are linear peptides with a reversal in chain polarity. The discovery of pseudovibriamides expands current knowledge of bacteria collective behavior. The establishment of reverse genetics will enable exploration of the ecological and biotechnological potential of Pseudovibrio bacteria. ClassificationBiological Sciences, Microbiology

Ecdysone coordinates plastic growth with robust pattern in the developing wing

Nogueira Alves, A. — 2020-12-16

Animals develop in unpredictable, variable environments. In response to environmental change some aspects of development adjust to generate plastic phenotypes. Other aspects of development, however, are buffered against environmental change to produce robust phenotypes. How organ development is coordinated to accommodate both plastic and robust developmental responses is poorly understood. Here, we demonstrate that the steroid hormone ecdysone coordinates both plasticity of organ size and robustness of organ pattern in the developing wings of the fruit fly Drosophila melanogaster. Using fed and starved larvae that lack prothoracic glands, which synthesise ecdysone, we show that nutrition regulates growth both via ecdysone and via an ecdysone-independent mechanism, while nutrition regulates patterning only via ecdysone. We then demonstrate that growth shows a graded response to ecdysone concentration, while patterning shows a threshold response. Collectively, these data support a model where nutritionally-regulated ecdysone fluctuations confer plasticity by regulating disc growth in response to basal ecdysone levels, and confers robustness by initiating patterning only once ecdysone peaks exceeds a threshold concentration. This could represent a generalizable mechanism through which hormones coordinate plastic growth with robust patterning in the face of environmental change.

Estradiol enhances ethanol stimulation of ventral tegmental area dopamine neuron firing without limiting ethanol inhibition onto those neurons

Lambeth, P. S. — 2020-12-22

Females can progress to alcohol and other substance use disorders more quickly than males. The ovarian hormone 17{beta}-estradiol (E2) contributes to sex differences observed in drug use and abuse and may be a principal driver of these differences. However, it is not entirely clear how E2 acts to affect processing of ethanol reward, and several brain regions and mechanisms are implicated. We sought to clarify the role of E2 in modulating the response of ventral tegmental area dopamine neurons to ethanol. To this end, we recorded spontaneous action potentials and inhibitory post synaptic currents from dopaminergic neurons in acute horizontal brain slices from ovariectomized (OVX) dopamine neuron reporter mice (Pitx3-eGFP) treated with either vehicle (VEH) or E2. On the basis of prior work, we hypothesized that E2 administration would cause dopamine cells from OVX+E2 animals to show a more substantial ethanol-induced increase in firing rate compared to control animals. Our data confirmed that ethanol stimulation of the firing rate of dopamine neurons from OVX+E2 mice was greater than that of OVX+VEH animals. Further, we hypothesized that the firing rate increase would be accompanied by a concomitant decrease in ethanol stimulated inhibition onto those same neurons. We found that although ethanol caused the expected increase in GABAA receptor-mediated synaptic inhibition in both groups, there was no difference in this response between OVX+E2 and OVX+VEH animals. Our findings lend additional support for the ability of E2 to enhance ventral tegmental area dopamine neuron responses to ethanol and suggest that this effect is not mediated by an E2-elicited suppression of synaptic inhibition.

A mRNA-LNP vaccine against Dengue Virus elicits robust, serotype-specific immunity

Wollner, C. J. — 2021-01-06

Dengue virus (DENV) is the most common vector-borne viral disease with nearly 400 million worldwide infections each year concentrated in the tropical and subtropical regions of the world. Severe dengue complications are often associated with a secondary heterotypic infection of one of the four circulating serotypes. In this scenario, humoral immune responses targeting cross-reactive, poorly-neutralizing epitopes can lead to increased infectivity of susceptible cells via antibody-dependent enhancement (ADE). In this way, antibodies produced in response to infection or vaccination are capable of contributing to enhanced disease in subsequent infections. Currently, there are no available therapeutics to combat DENV disease, and there is an urgent need for a safe and efficacious vaccine. Here, we developed a nucleotide-modified mRNA vaccine encoding for the membrane and envelope structural proteins from DENV serotype 1 encapsulated into lipid nanoparticles (prM/E mRNA-LNP). Vaccination of mice elicited robust antiviral immune responses comparable to viral infection with high levels of neutralizing antibody titers and antiviral CD4+ and CD8+ T cells. Immunocompromised AG129 mice vaccinated with the prM/E mRNA-LNP vaccine were protected from a lethal DENV challenge. Vaccination with either a wild-type vaccine, or a vaccine with mutations in the immunodominant fusion-loop epitope, elicited equivalent humoral and cell mediated immune responses. Neutralizing antibodies elicited by the vaccine were sufficient to protect against a lethal challenge. Both vaccine constructs demonstrated serotype specific immunity with minimal serum cross-reactivity and reduced ADE compared to a live DENV1 viral infection. IMPORTANCEWith 400 million worldwide infections each year, dengue is the most common vector-born viral disease. 40% of the worlds population is at risk with dengue experiencing consistent geographic spread over the years. With no therapeutics available and vaccines performing sub optimally, the need for an effective dengue vaccine is urgent. Here we develop and characterize a novel mRNA vaccine encoding for the dengue serotype 1 envelope and premembrane structural proteins that is delivered via a lipid nanoparticle. Our DENV1 prM/E mRNA-LNP vaccine induces neutralizing antibody and cellular immune responses in immunocompetent mice and protects an immunocompromised mouse from a lethal DENV challenge. Existing antibodies against dengue can enhance subsequent infections via antibody-dependent enhancement. Importantly our vaccine only induced serotype specific immune responses and did not induce ADE.

Prime editing enables precise genome editing in mouse liver and retina

Jang, H. — 2021-01-08

Prime editing can induce any small-sized genetic change without donor DNA or double strand breaks. However, it has not been investigated whether prime editing is possible in postnatal animals. Here we delivered prime editors 2 and 3 into a mouse model of hereditary tyrosinemia, a genetic liver disease, using hydrodynamic injection, which corrected the disease-causing mutation and rescued the phenotype. We also achieved prime editing in the retina and retina pigment epithelium in wild-type mice by delivering prime editor 3 using trans-splicing adeno-associated virus. Deep sequencing showed that unintended edits at or near the target site or off-target effects were not detectable except for low levels (0% to 1.2%) of indels when PE3, but not PE2, was used. Our study suggests that precise, prime editor-mediated genome editing is possible in somatic cells of adult animals.

Type IV competence pili in Streptococcus pneumoniae are highly dynamic structures that retract to promote DNA uptake

Lam, T. — 2021-01-18

The competence pili of transformable Gram-positive species form a subset of the diverse and widespread class of extracellular filamentous organelles known as type IV pili (T4P). In Gram-negative bacteria, T4P act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether T4P in Gram-positive species exhibit this same dynamic activity, and their mechanism of action for DNA uptake remains unclear. They are hypothesized to either (1) passively form transient cavities in the cell wall to facilitate DNA passage, (2) act as static adhesins to enrich DNA near the cell surface for subsequent uptake by membrane-embedded transporters, or (3) play an active role in translocating bound DNA via their dynamic activity. Here, using a recently described pilus labeling approach, we demonstrate that pneumococcal competence pili are highly dynamic structures that rapidly extend and retract from the cell surface. By labeling ComGC with bulky adducts, we further demonstrate that pilus retraction is essential for natural transformation. Together, our results indicate that Gram-positive type IV competence pili are dynamic and retractile structures that play an active role in DNA uptake. Short summaryCompetent pneumococci kill non-competent cells on contact. Retractable DNA-binding fibers in the class of type IV pili may provide a key tool for retrieving DNA segments from cell wreckage for internalization and recombination.

Universal DNA methylation age across mammalian tissues

Lu, A. T. — 2021-01-19

Aging is often perceived as a degenerative process resulting from random accrual of cellular damage over time. Despite this, age can be accurately estimated by epigenetic clocks based on DNA methylation profiles from almost any tissue of the body. Since such pan-tissue epigenetic clocks have been successfully developed for several different species, we hypothesized that one can build pan-mammalian clocks that measure age in all mammalian species. To address this, we generated data using 11,754 methylation arrays, each profiling up to 36 thousand cytosines in highly-conserved stretches of DNA, from 59 tissue-types derived from 185 mammalian species. From these methylation profiles, we constructed three age predictors, each with a single mathematical formula, termed universal pan-mammalian clocks that are accurate in estimating the age (r>0.96) of any mammalian tissue. Deviations between epigenetic age and chronological age relate to mortality risk in humans, mutations that affect the somatotropic axis in mice, and caloric restriction. We characterized specific cytosines, whose methylation levels change with age across most mammalian species. These cytosines are greatly enriched in polycomb repressive complex 2-binding sites, are located in regions that gradually lose chromatin accessibility with age and are proximal to genes that play a role in mammalian development, cancer, human obesity, and human longevity. Collectively, these results support the notion that aging is indeed evolutionarily conserved and coupled to developmental processes across all mammalian species - a notion that was long-debated without the benefit of this new compelling evidence. SUMMARYThis study identifies and characterizes evolutionarily conserved cytosines implicated in the aging process across mammals and establishes pan mammalian epigenetic clocks.

Shape Recovery of Deformed Biomolecular Droplets: Dependence on Condensate Viscoelasticity

Zhou, H.-X. — 2021-01-21

A theoretical study on the shape dynamics of phase-separated biomolecular droplets is presented, highlighting the importance of condensate viscoelasticity. Previous studies on shape dynamics have modeled biomolecular condensates as purely viscous, but recent data have shown them to be viscoelastic. Here we present an exact analytical solution for the shape recovery dynamics of deformed biomolecular droplets. The shape recovery of viscous droplets has an exponential time dependence, with the time constant given by the "viscocapillary" ratio, i.e., viscosity over interfacial tension. In contrast, the shape recovery dynamics of viscoelastic droplets is multi-exponential, with shear relaxation yielding additional time constants. During shape recovery, viscoelastic droplets exhibit shear thickening (increase in apparent viscosity) at fast shear relaxation rates but shear thinning (decrease in apparent viscosity) at slow shear relaxation rates. These results highlight the importance of viscoelasticity and expand our understanding of how material properties affect condensate dynamics in general, including aging.

TRPM7 is critical for short-term synaptic depression by regulating synaptic vesicle endocytosis

Jiang, Z. — 2021-01-25

TRPM7 contributes to a variety of physiological and pathological processes in many tissues and cells. With a widespread distribution in the nervous system, TRPM7 is involved in animal behaviors and neuronal death induced by ischemia. However, the physiological role of TRPM7 in CNS neuron remains unclear. Here, we identify endocytic defects in neuroendocrine cells and neurons from TRPM7 knockout (KO) mice, indicating a role of TRPM7 in synaptic vesicle endocytosis. Our experiments further pinpoint the importance of TRPM7 as an ion channel in synaptic vesicle endocytosis. Ca2+ imaging detects a defect in presynaptic Ca2+ dynamics in TRPM7 KO neuron, suggesting an importance of Ca2+ influx via TRPM7 in synaptic vesicle endocytosis. Moreover, the short-term depression is enhanced in both excitatory and inhibitory synaptic transmission from TRPM7 KO mice. Taking together, our data suggests that Ca2+ influx via TRPM7 may be critical for short-term plasticity of synaptic strength by regulating synaptic vesicle endocytosis in neurons.

UNC-2 CaV2 channel localization at presynaptic active zones depends on UNC-10/RIM and SYD-2/Liprin-α in Caenorhabditis elegans.

Oh, K. H. — 2021-01-27

Presynaptic active zone proteins couple calcium influx with synaptic vesicle exocytosis. However, the control of presynaptic calcium channel clustering by active zone proteins is not completely understood. In a C. elegans forward genetic screen, we find that UNC-10/RIM (Rab3-interacting molecule) and SYD-2/Liprin- regulate presynaptic clustering of UNC-2, the CaV2 channel ortholog. We further quantitatively analyzed live animals using endogenously GFP-tagged UNC-2 and active zone components. Consistent with the interaction between RIM and CaV2 in mammals, the intensity and number of UNC-2 channel clusters at presynaptic terminals were greatly reduced in unc-10 mutant animals. To understand how SYD-2 regulates presynaptic UNC-2 channel clustering, we analyzed presynaptic localization of endogenous SYD-2, UNC-10, RIMB-1/RIM-BP (RIM binding protein), and ELKS-1. Our analysis revealed that while SYD-2 is the most critical for active zone assembly, loss of SYD-2 function does not completely abolish presynaptic localization of UNC-10, RIMB-1, and ELKS-1, suggesting an existence of SYD-2-independent active zone assembly. UNC-2 localization analysis in double and triple mutants of active zone components show that SYD-2 promotes UNC-2 clustering by partially controlling UNC-10 localization, and ELKS-1 and RIMB-1 also contribute to UNC-2 channel clustering. In addition, we find that core active zone proteins are unequal in their abundance. While the abundance of UNC-10 at the active zone is comparable to UNC-2, SYD-2 and ELKS-1 are twice more and RIMB-1 four times more abundant than UNC-2. Together our data show that UNC-10, SYD-2, RIMB-1, and ELKS-1 control presynaptic UNC-2 channel clustering in redundant yet distinct manners. Significance StatementPrecise control of neurotransmission is dependent on the tight coupling of the calcium influx through voltage-gated calcium channels (VGCCs) to the exocytosis machinery at the presynaptic active zones. However, how these VGCCs are tethered to the active zone is incompletely understood. To understand the mechanism of presynaptic VGCC localization, we performed a C. elegans forward genetic screen and quantitatively analyzed endogenous active zones and presynaptic VGCCs. In addition to RIM (Rab3-interacting molecule), our study finds that SYD-2/Liprin- is critical for presynaptic localization of VGCCs. Yet, the loss of SYD-2, the master active zone scaffolding protein, does not completely abolish the presynaptic localization of the VGCC, showing that the active zone is a resilient structure assembled by redundant mechanisms.

Single calcium channel nanodomains drive presynaptic calcium entry at individual lamprey reticulospinal presynaptic terminals

Ramachandran, S. — 2021-02-02

Synchronous neurotransmission is central to efficient information transfer in neural circuits, requiring precise coupling between action potentials, Ca2+ entry and neurotransmitter release. However, determinations of Ca2+ requirements for release, which may originate from entry through single voltage-gated Ca2+ channels, remain largely unexplored in simple active zone synapses common in the nervous system. Understanding these requirements is key to addressing Ca2+ channel and synaptic dysfunction underlying numerous neurological and neuropsychiatric disorders. Here, we present single channel analysis of evoked active zone Ca2+ entry, using cell-attached patch clamp and lattice light sheet microscopy over active zones at single central lamprey reticulospinal presynaptic terminals. Our findings show a small pool (mean of 23) of Ca2+ channels at each terminal, comprising subtypes N-type (CaV2.2), P/Q-type (CaV2.1) and R-type (CaV2.3), available to gate neurotransmitter release. Significantly, of this pool only 1-6 (mean of 4) channels open upon depolarization. High temporal fidelity lattice light sheet imaging reveals AP-evoked Ca2+ transients exhibiting quantal amplitude variations between action potentials and stochastic variation of precise locations of Ca2+ entry within the active zone. Further, Ca2+ channel numbers at each active zone correlate to the number of presynaptic primed synaptic vesicles. Together, our findings indicate 1:1 association of Ca2+ channels with primed vesicles, suggesting Ca2+ entry via as few as one channel may trigger neurotransmitter release.

Knockdown of Dnmt1 links Gene body DNA methylation to regulation of gene expression and maternal-zygotic transition in the wasp Nasonia

Arsala, D. — 2021-02-02

Gene body methylation (GBM) is an ancestral aspect of DNA methylation (Sarda, Zeng, Hunt, & Yi, 2012; Yi, 2012; Zemach, McDaniel, Silva, & Zilberman, 2010) whose role in development has been obscured by the more prominent roles of promoter and CpG island methylation. The wasp Nasonia has little promoter and CpG island methylation, yet retains strong GBM (Park et al., 2011; Wang et al., 2013; Werren et al., 2010), making it an excellent model for elucidating the role of GBM. Here we show that Nasonia DNA methyl transferase 1a (Nv-Dnmt1a) knockdown leads to failures in cellularization and gastrulation of the embryo. Both of these disrupted events are hallmarks of the maternal-zygotic transition (MZT) in insects. Analysis of the embryonic transcriptome and methylome revealed strong reduction of GBM and widespread disruption of gene expression during embryogenesis after Nv-Dnmt1a knockdown. There was a strong correlation between loss of GBM and reduced gene expression in thousands of methylated loci, while affected unmethylated genes tended to be upregulated. We propose that reduced GBM and subsequent lower expression levels of methylated genes was the direct effect of Nv-Dnmt1 knockdown, and that this disruption led to widespread downstream dysregulation of MZT, and manifesting in developmental failure at gastrulation. Significance StatementThe importance of gene-body methylation (GBM) in development is unclear, due to the difficulty in teasing apart the effects of cis-regulatory methylation from those of GBM in vertebrate model systems. Unlike vertebrate models, the methylation machinery in the jewel wasp Nasonia vitripennis appears to exclusively mediate GBM, thus simplifying interpretation of the role of GBM in development. Knockdown of DNMT1 (Nv-Dnmt1a) in Nasonia leads to embryonic lethality, which we show is caused by a failure of cellularization and gastrulation. Nv-Dnmt1a knockdown resulted in a global loss of GBM in the embryo, which was strongly correlated with a down-regulation of gene expression. We propose that GBM facilitated by Nv-Dnmt1a is required for proper zygotic genome activation in the wasp.

Methylomic alteration in peripheral blood lymphocytes of prodromal stage and first-episode Chinese Han schizophrenia patients

Liu, Y. — 2021-02-09

BackgroundAlthough epigenetic dysregulation has long been proposed to promote the onset of schizophrenia, the landscape of the methylomic changes across the whole genome is yet established. MethodsUsing Infinium Human Methylation 850 BeadChip Array and MethylTarget sequencing method, we investigated the genome-wide methylation profiles and further validated methylation profiles of target genes in peripheral blood lymphocytes between individuals with psychosis risk syndrome (PRS), patients with first-episode schizophrenia (FES) and healthy controls (HC) in Chinese Han population. ResultsWe detected 372 sites between psychosis risk syndrome (PRS) and healthy controls (HC), which increased to 460 sites in first-episode schizophrenia (FES) with 207 sites shared. Both PRS and FES featured profound hypomethylation within gene body. Gene ontology and network annotation merged on loci enriched in disease associated signaling pathways (MAPK(Mitogen Activated Protein Kinases), Glutamatergic, GABAergic etc.). ConclusionsOur study implicated characteristic hypomethylation in both the discovery and validation cohorts in SYNGAP1, one of the frequently studied genes in neurodevelopmental disorders. This is the first methylome-wide association study between PRS and FES in Chinese Han population. Our findings provide potential biomarkers that can be used for future development of disease therapy and management.

Niche-guided tissue patterning by chemomechanical flow lithography

Newman, P. L. H. — 2021-02-11

Stem-cell derived tissue models are commonly cultured under globally-delivered stimuli that trigger histogenesis via self-organizing activity. However, the culture of such tissue models is prone to stochastic behavior, limiting the reproducibility of cellular composition and resulting in non-physiological architectures. To overcome these shortcomings, we developed a method for printing cell niche microenvironments with microstructured cues that mediate local histogenic processes, including mechanosensing and differentiation of selected cell types. Microstructured cues include independently tunable mechano-chemical properties, with conjugated peptides, proteins, and morphogens across a range of Youngs moduli. By rationally designing niches, we mediate the structure of tissues derived from stem-cell-progenitor sources, including a bone-fat assembly from stromal mesenchyme, and embryonic tissues derived from hiPSC. We show that microstructured cues can recapitulate mechano-chemical signals resembling early embryonic histogenesis. This outcome includes a role for niche mechanics in human embryonic organization, where soft niche mechanics bias markers of mesendodermal differentiation and epithelial-to-mesenchymal-transition (EMT), as well as a demonstration of a material-mediated morphogen signaling centers able to induce foci of mesenchymal and EMT differentiation. Thus, microstructured materials can mediate local histogenic processes to enhance the structure and composition of tissue models.

Tumor expressed CD95 causes suppression of anti-tumor activity of NK cells in a model of triple negative breast cancer

Qadir, A. S. — 2021-02-15

The apoptosis inducing receptor CD95/Fas has multiple tumorigenic activities. Stimulation by its cognate ligand CD95L on many cancer cells increases their growth, motility, ability to invade and/or their cancer stemness. Using genetically engineered mouse models of ovarian and liver cancer, we previously reported that deletion of CD95 in the tumor cells strongly reduced their ability to grow in vivo [1, 2]. Using a combination of immune-deficient and immune-competent mouse models, we now establish that loss of CD95 in metastatic triple negative breast cancer cells prevents tumor growth by modulating the immune landscape. CD95 deficient but not wild-type tumors barely grow in an immune-competent environment and show an increase in immune infiltrates into the tumor. This growth reduction is caused by NK cells and does not involve CD8+ T cells. On the other hand, in immune compromised mice CD95 k.o. cells are not growth inhibited, but they fail to form metastases. In summary, we demonstrate that in addition to its tumor and metastasis promoting activities, CD95 expression by tumor cells can exert immune suppressive activities providing a new target for immune therapy.

Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures

Shen, Z. — 2021-02-15

Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 {micro}M). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a "BL2 groove" that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

Macromolecular Regulators Have Matching Effects on the Phase Equilibrium and Interfacial Tension of Biomolecular Condensates

Mazarakos, K. — 2021-02-18

The interfacial tension of phase-separated biomolecular condensates affects their fusion and multiphase organization, and yet how this important property depends on the composition and interactions of the constituent macromolecules is poorly understood. Here we use molecular dynamics simulations to determine the interfacial tension and phase equilibrium of model condensate-forming systems. The model systems consist of binary mixtures of Lennard-Jones particles or chains of such particles. We refer to the two components as drivers and regulators; the former has stronger self-interactions and hence a higher critical temperature (Tc) for phase separation. In previous work, we have shown that, depending on the relative strengths of driver-regulator interactions and driver-driver interactions, regulators can either promote or suppress phase separation (i.e., increase or decrease Tc). Here we find that the effects of regulators on Tc quantitatively match the effects on interfacial tension ({gamma}). This important finding means that, when a condensate-forming system experiences a change in macromolecular composition or a change in intermolecular interactions (e.g., by mutation or posttranslational modification, or by variation in solvent conditions such as temperature, pH, or salt), the resulting change in Tc can be used to predict the change in{gamma} and vice versa. We also report initial results showing that disparity in intermolecular interactions drives multiphase coexistence. These findings provide much needed guidance for understanding how biomolecular condensates mediate cellular functions.

Open Natural Products Research: Curation and Dissemination of Biological Occurrences of Chemical Structures through Wikidata

Rutz, A. — 2021-03-01

Contemporary bioinformatic and chemoinformatic capabilities hold promise to reshape knowledge management, analysis and interpretation of data in natural products research. Currently, reliance on a disparate set of non-standardized, insular, and specialized databases presents a series of challenges for data access, both within the discipline and for integration and interoperability between related fields. The fundamental elements of exchange are referenced structure-organism pairs that establish relationships between distinct molecular structures and the living organisms from which they were identified. Consolidating and sharing such information via an open platform has strong transformative potential for natural products research and beyond. This is the ultimate goal of the newly established LOTUS initiative, which has now completed the first steps toward the harmonization, curation, validation and open dissemination of 750,000+ referenced structure-organism pairs. LOTUS data is hosted on Wikidata and regularly mirrored on https://lotus.naturalproducts.net. Data sharing within the Wikidata framework broadens data access and interoperability, opening new possibilities for community curation and evolving publication models. Furthermore, embedding LOTUS data into the vast Wikidata knowledge graph will facilitate new biological and chemical insights. The LOTUS initiative represents an important advancement in the design and deployment of a comprehensive and collaborative natural products knowledge base. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/433265v3_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@184136borg.highwire.dtl.DTLVardef@16e0d6org.highwire.dtl.DTLVardef@30ae1org.highwire.dtl.DTLVardef@1bf4825_HPS_FORMAT_FIGEXP M_FIG C_FIG

A comparative recombination analysis of human coronaviruses and implications for the SARS-CoV-2 pandemic

Pollett, S. — 2021-03-08

The SARS-CoV-2 pandemic prompts evaluation of recombination in human coronavirus (hCoV) evolution. We undertook recombination analyses of 158,118 public seasonal hCoV, SARS-CoV-1, SARS-CoV-2 and MERS-CoV genome sequences using the RDP4 software. We found moderate evidence for 8 SARS-CoV-2 recombination events, two of which involved the spike gene, and low evidence for one SARS-CoV-1 recombination event. Within MERS-CoV, 229E, OC43, NL63 and HKU1 datasets, we noted 7, 1, 9, 14, and 1 high-confidence recombination events, respectively. There was propensity for recombination breakpoints in structural genes, and recombination severely skewed the temporal structure of these data, especially for NL63 and OC43. Bayesian time-scaled analyses on recombinant-free data indicated the sampled diversity of seasonal CoVs emerged in the last 70 years, with 229E displaying continuous lineage replacements. These findings emphasize the importance of genomic based surveillance to detect recombination in SARS-CoV-2, particularly if recombination may lead to immune evasion.

Effect of Voluntary Adolescent Alcohol Consumption on Encoding of Decision-Related Variables in Prefrontal Cortex

Roitman, J. D. — 2021-03-10

The medial and orbitofrontal regions of prefrontal cortex (PFC) have been implicated in guiding optimal behavior and updating the economic value of rewards that result from choice behaviors. Both regions mature through adolescence into early adulthood and are thus vulnerable to exposure to neurotoxins, such as alcohol, during this critical developmental window. We sought to examine how voluntary alcohol consumption during adolescence would alter long-term PFC function and subsequent decision-making behavior in adulthood. Male and female rats were given adolescent intermittent ethanol (AIE) exposure to provide voluntary access to alcohol during the period of PFC maturation. In adulthood, we assessed the long-term effects on decision-making behavior using a risk task in adulthood, while concurrently recording neural activity in orbitofrontal cortex (OFC) and medial prefrontal cortex (mPFC). While control animals preferences for risky rewards increased with the likelihood of their delivery, AIE animals showed an overall reduction in their preferences for the risky option with higher levels of alcohol consumption, suggesting reduced discriminability of uncertain rewards and a shift away from the potential for reward omission. During task performance, neurons in mPFC and OFC responded to events (lever press, reward delivery). In mPFC, neurons with phasic increases at the time of lever press showed a reversal from larger elevations for risky presses in control animals to larger elevations for certain presses as prior alcohol consumption increased. Neurons in mPFC generally showed less discrimination of reward outcome with increased alcohol consumption as well. In OFC, responses to lever press were largely unaffected by AIE exposure. However, encoding of reward size in OFC showed differential effects in males and females. With higher alcohol consumption in males, OFC neurons showed largest excursions from baseline activity in response to largest reward, and smallest excursions for reward omission. This discrimination was reduced as prior alcohol consumption increased. In females, neurons with increased reward-activity, showed an overall higher level of activity due to stronger responses to certain rewards that were selected more frequently. Collectively the results show diminished capacity of PFC to encode decision-related elements to guide adaptive behavior and further clarify the lasting impact of adolescent alcohol use on neural function and behavior, even in the absence of continued use.

A role for the Erk MAPK pathway in modulating SAX-7/L1CAM-dependent locomotion in Caenorhabditis elegans

Moseley-Alldredge, M. — 2021-03-09

L1CAMs are immunoglobulin cell adhesion molecules that play important roles in the development and function of the nervous system. In addition to being associated with autism and schizophrenia spectrum disorders, impaired L1CAM function also underlies the X-linked L1 syndrome, which encompasses a group of neurological conditions, including spastic paraplegia and congenital hydrocephalus. Previous studies on both vertebrate and invertebrate L1CAMs established conserved roles that include axon guidance, dendrite morphogenesis, synapse development, and maintenance of neural architecture. We previously identified a genetic interaction between the C. elegans L1CAM encoded by the sax-7 gene and RAB-3, a GTPase that functions in synaptic neurotransmission; rab-3; sax-7 animals exhibit synthetic locomotion abnormalities and neuronal dysfunction. In this study, we examine the significance of this genetic interaction and show that this synergism also occurs when loss of SAX-7 is combined with mutants of other genes encoding key players of the synaptic vesicle cycle. In contrast, sax-7 does not interact with genes that function in synaptogenesis. These findings suggest a post-developmental role for sax-7 in the regulation of synaptic activity. To further assess this possibility, we conducted electrophysiological recordings and ultrastructural analyses at neuromuscular junctions. Lastly, we performed a forward genetic screen for suppressors of the rab-3; sax-7 synthetic phenotypes, uncovering a role for the Mitogen-activated Protein Kinase (MAPK) pathway in promoting coordinated locomotion.

BEdeepoff: an in silico tool for off-target prediction of ABE and CBE base editors

Zhang, C. — 2021-03-15

Base editors, including adenine base editors (ABEs) and cytosine base editors (CBEs), are valuable tools for introducing point mutations, but they frequently induce unwanted off-target mutations. Here, we performed a high-throughput gRNA-target library screening to measure editing efficiencies at integrated genomic off-targets and obtained datasets of 48,632 and 52,429 off-targets for ABE and CBE, respectively. We used the datasets to train deep learning models, resulting in ABEdeepoff and CBEdeepoff which can predict editing efficiencies at off-targets. These tools are freely accessible via online web server http://www.deephf.com/#/bedeep.

BEdeepon: an in silico tool for prediction of base editor efficiencies and outcomes

Zhang, C. — 2021-03-15

Base editors enable direct conversion of one target base into another in a programmable manner, but conversion efficiencies vary dramatically among different targets. Here, we performed a high-throughput gRNA-target library screening to measure conversion efficiencies and outcome product frequencies at integrated genomic targets and obtained datasets of 60,615 and 73,303 targets for ABE and CBE, respectively. We used the datasets to train deep learning models, resulting in ABEdeepon and CBEdeepon which can predict on-target efficiencies and outcome sequence frequencies. The software is freely accessible via online web server http://www.deephf.com/#/bedeep/bedeepon.

Identification of Japanese Encephalitis Virus Genotype V and Other Mosquito-borne Viruses in Camp Humphreys, Republic of Korea, using Metagenomic Analysis

Sanborn, M. A. — 2021-03-16

Recent outbreaks of emerging and re-emerging viruses such as Zika, West Nile and Japanese encephalitis (JEV) viruses have shown that timely detection of novel arboviruses with epidemic potential is essential to mitigate human health risks. There have been rising concerns that an emergent JEV genotype (genotype V, GV) is circulating in Asia, against which the current US-FDA-approved JEV vaccine may not be efficacious. To ascertain if JEV GV and other arboviruses are circulating in East Asia, we conducted next-generation sequencing on 260 pools of Culex tritaeniorhynchus and Culex bitaeniorhynchus mosquitoes (6,540 specimens) collected at Camp Humphreys, Republic of Korea (ROK), from mid-May - October 2018. Metagenomic analysis demonstrated a highly abundant and diverse virome with correlates of health and ecological relevance. Additionally, two complete JEV GV genome sequences were obtained from separate mosquito pools, indicating that JEV GV is circulating in the Pyeongtaek area near Seoul, ROK. Retrospective sample and sequence analyses showed that JEV GV was also present in 2016 mosquito pools collected in Seoul, ROK. Sequence-based analysis of JEV GV indicates a divergent genotype that is the most distant from the GIII derived live attenuated SA14-14-2 vaccine strain. A GV E protein investigation and 3D modeling in context to SA14-14-2 indicated likely regions responsible for reduced antibody affinity, including clusters of significant amino acid changes at externally exposed domains. These data highlight the critical need for continued mosquito surveillance as a means of detecting and identifying emerging and re-emerging arboviruses of public health relevance. Importantly, our results emphasize recent concerns that there may be a possible shift in the circulating JEV genotype in East Asia and highlights the critical need for a vaccine proven to be efficacious against this re-emergent virus.

Differential CTCF binding in motor neurons and lymphocytes

Atasever Arslan, B. — 2021-03-19

Transcriptional regulation of protein-coding genes is a primary control mechanism of cellular function. Similarities in regulation of expression for select genes between lymphocytes and neurons have led to proposals that such genes may be useful biomarkers for some neurological disorders that can be monitored via patient lymphocyte populations. Examination of shared molecular mechanisms underlying neurogenesis and lymphocyte differentiation may give help to identify relevant pathways and suggest additional biomarkers in lymphocytes that are relevant to neurological disorders. In this study, we analysed similarities and conserved regions in several genes regulated by CCCTC-binding factor (CTCF) during lymphocyte and neuronal developmental stages. We performed epigenetic analysis of CTCF binding Trak1, Gabpa, Gabpb1, Gabpb2, Gfi1, Gfi1b gene loci at T and B lymphocytes at different developmental stages, as well as in neural progenitor cells and motor neurons. Common and shared CTCF binding events at Trak1 gene suggest additional transcriptional regulatory factors that control Trak1 gene expression levels differ in neurons and lymphocytes. Gabpb1 gene includes a common CTCF binding site shared with neurons and lymphocytes. Correlation of CTCF binding analysis and gene expression profile suggests that CTCF binding plays a role in epigenetic regulation of Gabpb1 gene. In contrast, while Gfi1a gene is phylogenetically well-conserved and CTCF sites are occupied in lymphocytes, there are no CTCF binding occupied in neurons and neural progenitor cells. Low expression levels of Gfi1s in neurons indicate that regulation of this gene is CTCF-independent in neurons. Although Gfi1b is highly homologous to Gfi1, differences in expression levels suggest that Gfi1b is critical for both lymphogenesis and neurogenesis. Neurons and lymphocytes have multiple common CTCF binding sites in the Gfi1b gene, although lineage specific transcriptional regulators that play a role in their different expression levels still need to be identified. The partial overlap in CTCF regulatory sites for some genes in neurons and lymphocytes suggest that there may be markers that can exhibit parallel changes in these cells and serve as biomarkers.

Proteus mirabilis employs a contact-dependent killing system against competing Enterobacteriaceae

Kiani, D. — 2021-03-20

Many bacterial species encode systems for interference competition with other microorganisms. Some systems are effective without contact (e.g. through secretion of toxins), while other systems (e.g. Type VI secretion system (T6SS)) require direct contact between cells. Here, we provide the initial characterization of a novel contact-dependent competition system for Proteus mirabilis. In neonatal mice, a commensal P. mirabilis strain apparently eliminated commensal Escherichia coli. We replicated the phenotype in vitro and showed that P. mirabilis efficiently reduced viability of several Enterobacteriaceae species, but not Gram-positive species or yeast cells. Importantly, P. mirabilis strains isolated from humans also killed E. coli. Reduction of viability occurred from early stationary phase to 24h of culture and was observed in shaking liquid media as well as on solid media. Killing required contact, but was independent of T6SS, the only contact-dependent killing system described for P. mirabilis. Expression of the killing system was regulated by osmolarity and components secreted into the supernatant. Stationary phase P. mirabilis culture supernatant itself did not kill but was sufficient to induce killing in an exponentially growing co-culture. In contrast, killing was largely prevented in media with low osmolarity. In summary, we provide the initial characterization of a potentially novel interbacterial competition system encoded in P. mirabilis. IMPORTANCEThe study of bacterial competition systems has received significant attention in recent years. These systems collectively shape the composition of complex ecosystems like the mammalian gut. They are also being explored as narrow-spectrum alternatives to specifically eliminate problematic pathogenic species. However, many competition systems that effectively work in vitro do not show strong phenotypes in the gut. Our study was informed by an observation in infant mice. Further in vitro studies confirmed that P. mirabilis was able to kill several Enterobacteriaceae species. This killing system is novel for P. mirabilis and might represent a new function of a known system or even a novel system, as the observed characteristics do not fit with described contact-dependent competition systems. Competition systems are frequently present in multiple Enterobacteriaceae species. If present or transferred into a probiotic, it might be used in the future to reduce blooms of pathogenic Enterobacteriaceae associated with disease.

Refinement of corticospinal neuron activity during skilled motor learning

Serradj, N. — 2021-03-22

The learning of motor skills relies on plasticity of the primary motor cortex as task acquisition drives the remodeling of cortical motor networks1,2. Large scale cortical remodeling of evoked motor outputs occurs in response to the learning of skilled, corticospinal-dependent behavior, but not simple, unskilled tasks1. Here we determine the response of corticospinal neurons to both skilled and unskilled motor training and assess the role of corticospinal neuron activity in the execution of the trained behaviors. Using in vivo calcium imaging, we found temporal coding of corticospinal activity coincided with the development of skilled, but not unskilled, motor expertise. Animals that failed to learn our skilled, precision isometric pull task exhibited a limited repertoire of dynamic movements and reduced network modulation. Transection of the corticospinal tract and optogenetic regulation of corticospinal activity show the necessity for patterned corticospinal network activity in the execution of skilled, but not unskilled, movement. We reveal a critical role for corticospinal network modulation in the learning and execution of skilled motor movements. The integrity of the corticospinal tract is essential to the recovery of dexterous movement after central nervous system injuries and these findings should help to shape translational approaches to motor recovery.

Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

van der Stel, A.-X. — 2021-03-31

Free L-tryptophan (L-Trp) induces the expression of the Escherichia coli tryptophanase operon, leading to the production of indole from L-Trp. Tryptophanase operon expression is controlled via a mechanism involving the tryptophan-dependent stalling of ribosomes engaged in translation of tnaC, a leader sequence upstream of tnaA that encodes a 24-residue peptide functioning as a sensor for L-Trp. Although extensive biochemical characterization has revealed the elements of the TnaC peptide and the ribosome that are responsible for translational arrest, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a variant of TnaC (R23F) in which stalling by L-Trp is enhanced because of reduced cleavage of TnaC(R23F)-peptidyl-tRNA. In contrast to previous data originated from lower resolution structural studies, we show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo tryptophan-dependent termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. In addition, we show that the conformation of the L-Trp binding site is not altered by the R23F mutation. This leads us to propose a model in which rates of TnaC-peptidyl-tRNA cleavage by release factor and binding of the L-Trp ligand to the translating ribosome determine the tryptophan sensitivity of the wild-type and mutant TnaC variants. Thus, our study reveals a strategy whereby a nascent peptide assists the bacterial ribosome in sensing a small metabolite.

E2F/Dp inactivation in fat body cells triggers systemic metabolic changes

Zappia, M. P. — 2021-04-09

The E2F transcription factors play a critical role in controlling cell fate. In Drosophila, the inactivation of E2F in either muscle or fat body results in lethality, suggesting an essential function for E2F in these tissues. However, the cellular and organismal consequences of inactivating E2F in these tissues are not fully understood. Here, we show that the E2F loss exerts both tissue-intrinsic and systemic effects. The proteomic profiling of E2F-deficient muscle and fat body revealed that E2F regulates carbohydrate metabolism, a conclusion further supported by metabolomic profiling. Intriguingly, animals with E2F-deficient fat body had a lower level of circulating trehalose and reduced storage of fat. Strikingly, a sugar supplement was sufficient to restore both trehalose and fat levels, and subsequently, rescued animal lethality. Collectively, our data highlight the unexpected complexity of E2F mutant phenotype, which is a result of combining both tissue-specific and systemic changes that contribute to animal development.

Stem cell-laden hydrogel bioink for generation of high resolution and fidelity engineered tissues with complex geometries

Jeon, O. — 2021-04-15

Recently, 3D bioprinting has been explored as a promising technology for biomedical applications with the potential to create complex structures with precise features. Cell encapsulated hydrogels composed of materials such as gelatin, collagen, hyaluronic acid, alginate and polyethylene glycol have been widely used as bioinks for 3D bioprinting. However, since most hydrogel-based bioinks may not allow rapid stabilization immediately after 3D bioprinting, achieving high resolution and fidelity to the intended architecture is a common challenge in 3D bioprinting of hydrogels. In this study, we have utilized shear-thinning and self-healing ionically crosslinked oxidized and methacrylated alginates (OMAs) as a bioink, which can be rapidly gelled by its self-healing property after bioprinting and further stabilized via secondary crosslinking. It was successfully demonstrated that stem cell-laden calcium-crosslinked OMA hydrogels can be bioprinted into complicated 3D tissue structures with both high resolution and fidelity. Additional photocrosslinking enables long-term culture of 3D bioprinted constructs for formation of functional tissue by differentiation of encapsulated human mesenchymal stem cells.

Shear Relaxation Governs Dynamic Processes of Biomolecular Condensates

Ghosh, A. — 2021-04-19

Phase-separated biomolecular condensates must respond agilely to biochemical and environmental cues in performing their wide-ranging cellular functions, but our understanding of condensate dynamics is lagging. Ample evidence now indicates biomolecular condensates as viscoelastic fluids, where shear stress relaxes at a finite rate, not instantaneously as in viscous liquids. Yet the fusion dynamics of condensate droplets has only been modeled based on viscous liquids, with fusion time given by the viscocapillary ratio (viscosity over interfacial tension). Here we used optically trapped polystyrene beads to measure the viscous and elastic moduli and the interfacial tensions of four types of droplets. Our results challenge the viscocapillary model, and reveal that the relaxation of shear stress governs fusion and other dynamic processes of condensates.

Spatially resolved analysis of Pseudomonas aeruginosa biofilm proteomes measured by laser ablation sample transfer

Hanley, L. — 2021-04-19

Heterogeneity in the distribution of nutrients and O2 gradients during biofilm growth gives rise to changes in phenotype. There has been long term interest in identifying spatial differences during biofilm development including clues that identify chemical heterogeneity. Laser ablation sample transfer (LAST) allows site-specific sampling combined with label free proteomics to distinguish radially and axially resolved proteomes for Pseudomonas aeruginosa biofilms. Specifically, differential protein abundances on oxic vs. anoxic regions of a biofilm was observed by combining LAST with bottom up proteomics. This study reveals active metabolism in the anoxic region of the biofilm with respect to the oxic region in P. aeruginosa, an aerobe by nature. Protein abundance data related to cellular acclimations to chemical gradients include identification of glucose catabolizing proteins, high abundance of proteins from arginine and polyamine metabolism, and proteins that could also support virulence and environmental stress mediation on the anoxic region. Finally, this methodology requires only a few mm2 of biofilm area to identify hundreds of proteins.

Deep Mutagenesis of a Transporter for Uptake of a Non-Native Substrate Identifies Conformationally Dynamic Regions

Young, H. J. — 2021-04-19

The serotonin transporter, SERT, catalyzes serotonin reuptake at the synapse to terminate neurotransmission via an alternating access mechanism, and SERT inhibitors are the most widely prescribed antidepressants. Here, deep mutagenesis is used to determine the effects of nearly all amino acid substitutions on human SERT surface expression and transport of the fluorescent substrate APP+, identifying many mutations that enhance APP+ import. Comprehensive simulations of the entire ion-coupled import process reveal that while binding of the native substrate, serotonin, reduces free energy barriers between conformational states to promote SERT dynamics, the conformational free energy landscape in the presence of APP+ instead resembles Na+ bound-SERT, with a higher free energy barrier for transitioning to an inward-facing state. The deep mutational scan for SERT-catalyzed import of APP+ finds mutations that promote the necessary conformational changes that would otherwise be facilitated by the native substrate. Indeed, hundreds of gain-of-function mutations for APP+ import are found along the permeation pathway, most notably mutations that favor the formation of a solvent-exposed intracellular vestibule. The mutagenesis data support the simulated mechanism in which the neurotransmitter and a symported sodium share a common cytosolic exit pathway to achieve coupling. Furthermore, the mutational landscape for SERT surface expression, which likely filters out misfolded sequences, reveals that residues along the permeation pathway are mutationally tolerant, providing plausible evolutionary pathways for changes in transporter properties while maintaining folded structure.

Computer-Vision Stabilized Intravital Imaging Reveals Lung Capillary Neutrophil Dynamics Crucial for Lung Host-Defense Function

Tsukasaki, Y. — 2021-04-19

Polymorphonuclear neutrophils (PMN) are highly dynamic innate immune cells which are essential for lung host defense. However, in vivo intravital imaging in moving organs such as the lung remains challenging due to motion artifacts. Here we describe a novel intravital imaging method with high-throughput analytical capability based on a computer vision stabilization algorithm, Computer-vision-Assisted STabilized intravital imaging (CASTii). The sub-micron precision of this approach enables analysis of compartmentalized intravital PMN dynamics. We quantified in real-time a novel patrolling function of lung intracapillary circulating PMN. We also describe the dynamics of intracapillary PMN pooling (marginated PMN pool) using direct imaging of PMNs. The pool was formed by repeated catch-and-release kinetics involving PMN deformation inside microvessels during the passage of PMNs in vessels. We observed rapid PMN recruitment into the lung tissue compartments from pooled PMNs in response to alveolar chemoattract exposure. In contrast, endotoxemia-induced intracapillary sequestration of PMN impaired PMN transmigration into the alveolar space and defective phagocytosis of live bacteria. Intravital imaging of PMN dynamics with CASTii provides fundamental insights into host-defense functions of lung capillary PMN.

Characterization of methylation signatures in spontaneous preterm birth

Brockway, H. M. — 2021-04-27

Preterm birth is a global public health crisis which results in significant neonatal and maternal mortality. Yet little is known regarding the molecular mechanisms of idiopathic spontaneous preterm birth, and we have few diagnostic markers for adequate assessment of placental development and function. Previous studies of placental pathology and our transcriptomics studies suggest a role for placental maturity in idiopathic spontaneous preterm birth. It is known that placental DNA methylation changes over gestation. We hypothesized that if placental hypermaturity is present in our samples, we would observe a unique idiopathic spontaneous preterm birth DNA methylation profile potentially driving the gene expression differences we previously identified in our placental samples. Our results indicate the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern suggesting hypermaturity. Only seven significant differentially methylated regions fitting the idiopathic spontaneous preterm birth specific (relative to the controls) profile were identified, indicating unusually high similarity in DNA methylation between idiopathic spontaneous preterm birth and term birth samples. We identified an additional 1,718 significantly methylated regions in our gestational age matched controls were the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern, again indicating a striking level of similarity between the idiopathic spontaneous preterm birth and term birth samples. Pathway analysis of these regions revealed differences in genes within the WNT and Cadherin signaling pathways, both of which are essential in placental development and maturation. Taken together, these data demonstrate that the idiopathic spontaneous preterm birth samples are molecularly more mature than expected given their respective gestational age which likely impacts birth timing.

Vitamin D Receptor Upregulates Tight Junction Protein Claudin-5 against Tumorigenesis

Zhang, Y.-g. — 2021-04-30

Background/ObjectiveTight junctions (TJs) are essential for barrier integrity, inflammation, and cancer. The TJ protein Claudin-5 in the epithelia forms paracellular barriers and pores for permeability. Vitamin D and the vitamin D receptor (VDR) play important roles in various cancers. Although VDR and Claudin-5 are all involved in colorectal cancer (CRC), it remains unclear if they are closely related or function independently. DesignUsing the human CRC database, we explored the correlation between VDR and Claudin-5. We then investigated the VDR regulation of Claudin-5 using VDR knockout (VDR-/-) and intestinal epithelial VDR knockout mice (VDR{Delta}IEC) with chemical-induced colon cancer and an epithelial VDR overexpression model. Human samples, organoids, and intestinal epithelial cells were used to determine the underlying mechanisms. ResultsIn human colon cancer, colonic VDR expression was low and was significantly correlated with a reduction of Claudin-5 mRNA and protein. In the colon of VDR-/- and VDR{Delta}IEC mice, deletion of VDR led to lower protein and mRNA levels of Claudin-5. Intestine permeability was increased in the AOM-DSS-induced VDR-/- colon cancer model. Lack of VDR and a reduction of Claudin-5 are associated with an increased number of tumors in the VDR-/- and VDR{Delta}IEC mice. Furthermore, gain and loss of function studies have identified CLDN-5 as a downstream target of the VDR signaling pathway. Epithelial VDR overexpression protected against the loss of Claudin 5 in response to intestinal inflammation ConclusionThis study advances the understanding of how VDR regulates intestinal barrier functions in tumorigenesis as a biomarker and potential treatment. A short summaryO_LIWhat is already known about this subject? O_LITight junction structures are essential for intestinal barrier integrity, inflammation, and cancer. C_LIO_LIVitamin D deficiency and the vitamin D receptor (VDR) play important roles in the development of colon cancer. C_LI C_LIO_LIWhat are the new findings? O_LIOur study is the first to link barrier function, a specific tight junction protein, and genetic susceptibility through intestinal epithelial VDR in human colorectal cancer. C_LIO_LIOur study fills an existing gap by characterizing the mechanism of intestinal epithelial VDR in regulating barrier functions through alterations in TJs in tumorigenesis. VDR is important for the maintenance of the physiological level of the TJ protein Claudin-5 in the colon. The CLDN-5 gene is a downstream target of the VDR signaling pathway. Lack of VDR led to a reduction of Claudin-5 in tumors, whereas enhancing VDR increased Claudin-5 to protect the intestinal epithelial cells from tumorigenesis. C_LIO_LIWe report fecal VDR reduction in a colon cancer model. This introduces the possibility for the identification of new biomarkers and therapeutic targets to restore VDR-dependent functions in CRC. C_LI C_LIO_LIHow might it impact on clinical practice in the foreseeable future O_LIDiagnosis of CRC considering VDR status C_LIO_LIBarrier: direct, indirect biomarkers C_LIO_LIIntestinal barriers in cancer prevention and treatment C_LI C_LI Barrier function and VDR are not only essential for the maintenance of intestinal homeostasis, but they are also critical for the development of chronic mucosal inflammation and cancer. This knowledge can be immediately used to develop intestinal VDR and Claudin-5 as clinical biomarkers for identifying patients who may benefit from currently available interventions and could also be used for the eventual development of novel strategies for the prevention and treatment of human CRC.

A multimodal study of a first episode psychosis cohort: potential markers of antipsychotic treatment resistance

Yang, K. — 2021-05-04

Treatment resistant (TR) psychosis is considered to be a significant cause of disability and functional impairment. Numerous efforts have been made to identify the clinical predictors of TR. However, the exploration of molecular and biological markers is still at an early stage. To understand the TR condition and identify potential molecular and biological markers, we analyzed demographic information, clinical data, structural brain imaging data, and molecular brain imaging data in 7 Tesla magnetic resonance spectroscopy, from a first episode psychosis cohort that includes 138 patients. Age, gender, race, smoking status, duration of illness, and antipsychotic dosages were controlled in the analyses. We found that TR patients had a younger age at onset, more hospitalizations, more severe negative symptoms, a significant reduction in the volumes of the hippocampus (HP) and superior frontal gyrus (SFG), and a significant reduction in glutathione (GSH) levels in the anterior cingulate cortex (ACC), when compared to non-TR patients. The combination of multiple markers provided a better classification between TR and non-TR patients compared to any individual marker. Our study shows that ACC GSH, HP and SFG volumes, and age at onset could potentially be trait biomarkers for TR diagnosis, while hospitalization and negative symptoms could be used to evaluate the progression of the disease. Multimodal cohorts are essential in obtaining a comprehensive understanding of brain disorders.

Identification of organ-specific transcriptomic shifts in the vasculature during systemic inflammation using TrendCatcher

Rehman, J. — 2021-05-04

Studying temporal gene expression shifts during disease progression provides important insights into the biological mechanisms that distinguish adaptive and maladaptive responses. Existing tools for the analysis of time course transcriptomic data are not designed to optimally identify distinct temporal patterns when analyzing dynamic differentially expressed genes (DDEGs). Moreover, there is a lack of methods to assess and visualize the temporal progression of biological pathways mapped from time course transcriptomic datasets. In this study, we developed an open-source R package TrendCatcher (https://github.com/jaleesr/TrendCatcher), which applies the smoothing spline ANOVA model and break point searching strategy to identify and visualize distinct dynamic transcriptional gene signatures and biological processes from longitudinal datasets. We used TrendCatcher to perform a systematic temporal analysis of COVID-19 peripheral blood transcriptomes, including bulk RNA-seq and scRNA-seq time course data. TrendCatcher uncovered the early and persistent activation of neutrophils and coagulation pathways as well as impaired type I interferon (IFN-I) signaling in circulating cells as a hallmark of patients who progressed to severe COVID-19, whereas no such patterns were identified in individuals receiving SARS- CoV-2 vaccinations or patients with mild COVID-19. These results underscore the importance of systematic temporal analysis to identify early biomarkers and possible pathogenic therapeutic targets.

Genome-wide admixture mapping of eGFR and CKD identify European and African ancestry-of-origin loci in U.S. Hispanics/Latinos

Horimoto, A. R. V. R. — 2021-05-06

BackgroundAdmixture mapping is a powerful approach for gene mapping of complex traits that leverages the diverse genetic ancestry in populations with recent admixture such as U.S. Hispanics/Latinos (HL), who have increased risk of chronic kidney disease (CKD). MethodsGenome-wide admixture mapping was performed for CKD and estimated glomerular filtration rate (eGFR) in a sample of 12,601 participants from the Hispanic Community Health Study/Study of Latinos, with validation in a sample of 8191 African Americans from the Womens Health Initiative (WHI). ResultsThree novel ancestry-of-origin loci were identified on chromosomes 2, 14 and 15 for CKD and eGFR. The chromosome 2 locus (2p16.3) consisted of two European ancestry regions encompassing the FSHR and NRXN1 genes, with European ancestry at this locus associated with increased risk for CKD. The chromosome 14 locus (14q32.2) located within the DLK1-DIO3 imprinted domain was driven by European ancestry, and was associated with lower eGFR. The chromosome 15 locus (15q13.3-14) included intronic variants of RYR3 and was within an African-specific genomic region that was associated with higher eGFR. These findings were compared to the conventional genome-wide association study that failed to identify significant associations in these regions. We validated the chromosome 14 and 15 loci for eGFR in the WHI African Americans. ConclusionsThis study provides evidence of shared ancestry-specific genomic regions influencing eGFR in HL and African Americans, and illustrates the potential for leveraging genetic ancestry in recently admixed populations for novel discovery of kidney trait loci.

Localization and RNAi-driven inhibition of a Brugia malayi encoded Interleukin-5 Receptor Binding protein

Mejia, R. — 2021-05-14

A molecule termed BmIL5Rbp (aka Bm8757) was identified from Brugia malayi filarial worms and found to competitively inhibit human IL-5 binding to its human receptor. After the expression and purification of a recombinant BmIL5Rbp and generation of BmIL5Rbp-specific rabbit antibody, we localized the molecule on B. malayi worms through immunohistochemistry and immunoelectron microscopy. RNA interference was used to inhibit BmIL5Rbp mRNA and protein production. BmIL5Rbp was shown to localize to the cuticle of Brugia malayi and to be released in their excretory/secretory products. RNAi inhibited BmIL5Rbp mRNA production by 33% and reduced the surface protein expression by [~]50% and suppressed the release of BmIL5Rbp in the excretory/secretory products. RNAi has been used successfully to knock down the mRNA and protein expression of BmIL5Rbp in the early larval stages of B. malayi and provided a proof-of-principle for the local inhibition of the human IL5 receptor. These findings provide evidence that a parasite encoded IL5R antagonist could be utilized therapeutically.

Epigenetic predictors of maximum lifespan and other life history traits in mammals

Li, C. Z. — 2021-05-18

Maximum lifespan of a species is the oldest that individuals can survive, reflecting the genetic limit of longevity in an ideal environment. Here we report methylation-based models that accurately predict maximum lifespan (r=0.89), gestational time (r=0.96), and age at sexual maturity (r=0.87), using cytosine methylation patterns collected from over 12,000 samples derived from 192 mammalian species. Our epigenetic maximum lifespan predictor corroborated the extended lifespan in growth hormone receptor knockout mice and rapamycin treated mice. Across dog breeds, epigenetic maximum lifespan correlates positively with breed lifespan but negatively with breed size. Lifespan-related cytosines are located in transcriptional regulatory regions, such as bivalent chromatin promoters and polycomb-repressed regions, which were hypomethylated in long-lived species. The epigenetic estimators of maximum lifespan and other life history traits will be useful for characterizing understudied species and for identifying interventions that extend lifespan.

Synaptic Plasticity in the Agranular Insular Cortex Predicts Escalated Ethanol Consumption

Shillinglaw, J. E. — 2021-05-18

The Agranular Insular Cortex (AIC) is implicated in alcohol use disorder and pharmacologically relevant concentrations of acute ethanol inhibit N-methyl-D-aspartate receptor (NMDAR)-mediated glutamatergic synaptic transmission and plasticity onto layer 2/3 AIC pyramidal neurons. However, it is not known whether the actions of ethanol on glutamatergic synapses are means by which chronic ethanol alters mechanisms of learning and memory in AIC as alcohol drinking transitions from controlled to problematic. We utilized the chronic intermittent ethanol (CIE) vapor model of ethanol exposure in adult male mice, alone or in combination with voluntary ethanol consumption, to determine whether glutamatergic synapses on layer 2/3 AIC pyramidal neurons are differentially regulated by different durations and intensities of chronic ethanol exposure. We observed evidence of both ethanol- and age-related metaplasticity of AIC layer 2/3 glutamatergic synapses, as only young adult, ethanol-naive mice exhibited NMDAR-dependent long term depression ex vivo. Our findings also indicated that voluntary ethanol consumption alone can elicit glutamatergic plasticity in vivo. We found that the ratio of NMDAR- to AMPAR-mediated postsynaptic currents was reduced not only in CIE-treated, but also in air-treated, chronically drinking mice relative to ethanol-naive controls. Furthermore, lower NMDA/AMPA ratios were predictive of greater escalation of ethanol consumption. These findings suggest that even moderate exposure to ethanol may elicit plasticity in the agranular insular cortex that contributes to the progression toward uncontrolled drinking.

Whole-ExM: Expansion microscopy imaging of all anatomical structures of whole larval zebrafish

Sim, J. — 2021-05-18

Nanoscale resolution imaging of whole vertebrates is required for a systematic understanding of human diseases, but this has yet to be realized. Expansion microscopy (ExM) is an attractive option for achieving this goal, but the expansion of whole vertebrates has not been demonstrated due to the difficulty of expanding hard body components. Here, we demonstrate whole-body ExM, which enables nanoscale resolution imaging of anatomical structures, proteins, and endogenous fluorescent proteins (FPs) of whole zebrafish larvae and mouse embryos by expanding them fourfold. We first show that post-digestion decalcification and digestion kinetics matching are critical steps in the expansion of whole vertebrates. Then, whole-body ExM is combined with the improved pan-protein labeling approach to demonstrate the three-dimensional super-resolution imaging of antibody- or FP-labeled structures and all major anatomical structures surrounding them. We also show that whole-body ExM enables visualization of the nanoscale details of neuronal structures across the entire body.

Measuring mitochondrial electron transfer complexes in previously frozen cardiac tissue from the offspring of sow: A model to assess exercise induced mitochondrial bioenergetics changes

Barrera, D. — 2021-06-09

Measuring the mitochondrial electron transfer complex (ETC) profile from previously frozen heart tissue samples from offspring born to an exercised sow provided descriptive data about exercise-induced mitochondrial biochemical changes in heart tissue from the offspring born to the exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis. SUMMARYPreparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

Removing Thermostat Distortions of Protein Dynamics in Constant-Temperature Molecular Dynamics Simulations

Hicks, A. — 2021-06-08

Molecular dynamics simulations are widely used to determine equilibrium and dynamic properties of proteins. Nearly all simulations nowadays are carried out at constant temperature, with a Langevin thermostat among the most widely used. Thermostats distort protein dynamics, but whether or how such distortions can be corrected has long been an open question. Here we show that constant-temperature simulations with a Langevin thermostat dilate protein dynamics and present a correction scheme to remove the dynamic distortions. Specifically, ns-scale time constants for overall rotation are dilated significantly but sub-ns time constants for internal motions are dilated modestly, while all motional amplitudes are unaffected. The correction scheme involves contraction of the time constants, with the contraction factor a linear function of the time constant to be corrected. The corrected dynamics of eight proteins are validated by NMR data for rotational diffusion and for backbone amide and side-chain methyl relaxation. The present work demonstrates that, even for complex systems like proteins with dynamics spanning multiple timescales, one can predict how thermostats distort protein dynamics and remove such distortions. The correction scheme will have wide applications, facilitating force-field parameterization and propelling simulations to be on par with NMR and other experimental techniques in determining dynamic properties of proteins. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/447619v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1aa72e8org.highwire.dtl.DTLVardef@15e0a4eorg.highwire.dtl.DTLVardef@f6c41borg.highwire.dtl.DTLVardef@c4e8e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cerebral hemodynamics: a mathematical model including autoregulation, baroreflex and extracranial peripheral circulation

Ambrosio Garcia, F. — 2021-06-11

Cerebral autoregulation, the physiological capability to regulate cerebral blood flow, may be assisted by short-term mean arterial pressure control via baroreflex, which, among several effects, modulates total peripheral resistance. It is unclear, however, whether the resistance of the head and neck vasculatures is also affected by baroreflex and whether these extracranial vessels assist autoregulation. Since sensing technologies such as functional Near-Infrared Spectroscopy and noninvasive intracranial pressure monitoring by strain gauge may be influenced by superficial tissue, it is clinically relevant to understand the relations between intracranial and extracranial hemodynamics. Therefore, we created an autoregulation model consisting of arteries and arterioles regulated by the intralumial pressure and microcirculation regulated by local blood flow. As the first critical step to quantify the signal deterioration introduced by the extracranial circulation on superficial sensors, the extracranial peripheral circulation of the head and neck and baroreflex regulation of the peripheral vasculature and of heart rate were also included. During simulations of a bout of acute hypotension, the model predicts a rapid return of cerebral blood flow to baseline levels and a prolonged suppression of the blood flow to the external carotid vasculature, in accordance with experimental evidence. The inclusion of peripheral control via baroreflex at the external carotid vasculature did not assist cerebral autoregulation, thus we raise the hypothesis that baroreflex may act on the head and neck vasculatures but this action has negligible effects on regulation of cerebral blood flow. When autoregulation is impaired, results suggest that the blood flow of the brain and of the head and neck present similar dynamics, while they are weakly coupled when autoregulation is intact. The model also provides a mechanistic explanation of the protection brought by cerebral autoregulation to the microvasculature and to the brain parenchyma. Our model forms the foundation for predicting the interference introduced by the superficial tissue to nonivasive sensors.

A secondary metabolite drives intraspecies antagonism in a gut symbiont that is inhibited by peptidoglycan acetylation

Özcam, M. — 2021-06-12

The mammalian microbiome encodes numerous secondary metabolite biosynthetic gene clusters, yet their role in microbe-microbe interactions is unclear. Here, we characterized two polyketide synthase gene clusters (fun and pks) in the gut symbiont Limosilactobacillus reuteri. The pks, but not the fun cluster, encodes antimicrobial activity. Forty-one out of 51 L. reuteri strains tested are sensitive to Pks products, which was independent of strains host origin. The sensitivity to Pks was also established in intraspecies competition experiments in gnotobiotic mice. Comparative genome analyses between Pks-resistant and sensitive strains identified an acyltransferase gene (act) that is unique to Pks-resistant strains. Subsequent cell wall analysis of the wild-type and the act mutant strains showed that Act acetylates cell wall components. The pks mutants lost their competitive advantage and act mutants lost their Pks resistance in vivo. Thus, our findings provide insight into how closely related gut symbionts can compete and co- exist in the gastrointestinal tract.

Structural basis for the context-specific action of classic peptidyl transferase inhibitors.

Syroegin, E. A. — 2021-06-18

Ribosome-targeting antibiotics serve both as powerful antimicrobials and as tools for studying the ribosome. The ribosomal catalytic site, the peptidyl transferase center (PTC), is targeted by a large number of various drugs. The classical and best-studied PTC-acting antibiotic chloramphenicol, as well as the newest clinically significant linezolid, were considered indiscriminate inhibitors of every round of peptide bond formation, presumably inhibiting protein synthesis by stalling ribosomes at every codon of every gene being translated. However, it was recently discovered that chloramphenicol or linezolid, and many other PTC-targeting drugs, preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms and structural bases that underlie this phenomenon of context-specific action of even the most basic ribosomal antibiotics, such as chloramphenicol, are unknown. Here we present high-resolution structures of ribosomal complexes, with or without chloramphenicol, carrying specific nascent peptides that support or negate the drug action. Our data suggest that specific amino acids in the nascent chains directly modulate the antibiotic affinity to the ribosome by either establishing specific interactions with the drug molecule or obstructing its placement in the binding site. The model that emerged from our studies rationalizes the critical importance of the penultimate residue of a growing peptide for the ability of the drug to stall translation and provides the first atomic-level understanding of context specificity of antibiotics that inhibit protein synthesis by acting upon the PTC.

A Machine Learning-Enabled Pipeline for Large-Scale Virtual Drug Screening

Gupta, A. — 2021-06-21

Virtual screening is receiving renewed attention in drug discovery, but progress is hampered by challenges on two fronts: handling the ever increasing sizes of libraries of drug-like compounds, and separating true positives from false positives. Here we developed a machine learning-enabled pipeline for large-scale virtual screening that promises breakthroughs on both fronts. By clustering compounds according to molecular properties and limited docking against a drug target, the full library was trimmed by 10-fold; the remaining compounds were then screened individually by docking; and finally a dense neural network was trained to classify the hits into true and false positives. As illustration, we screened for inhibitors against RPN11, the deubiquitinase subunit of the proteasome and a drug target for breast cancer. TOC Graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

EXC-4/CLIC, Gα, and Rho/Rac signaling regulate tubulogenesis in C. elegans

Arena, A. F. — 2021-06-21

The Rho-family of small GTPases, which play crucial roles in development and disease, are regulated by many signal-transduction cascades, including G-protein-coupled receptor (GPCR)-heterotrimeric G-protein (G/{beta}/{gamma}) pathways. Using genetic approaches in C. elegans we identified a new role for G and Rho/Rac signaling in cell outgrowth during tubulogenesis and show that the Chloride Intracellular Channel (CLIC) protein EXC-4 is an evolutionarily-conserved player in this pathway. The gene exc-4 was identified by its role in tubulogenesis of the excretory canal (ExCa) cell--a unicellular tube required for osmoregulation and fluid clearance. We identified an exc-4 loss-of-function allele that affects an evolutionarily conserved residue in the C-terminus. Using this mutant we identified genetic interactions between exc-4, G, and Rho-family GTPases, defining novel roles for G-encoding genes (gpa-12/G12/13, gpa-7/Gi, egl-30/Gq, gsa-1/Gs) and the Rho-family members ced-10/Rac and mig-2/RhoG in ExCa outgrowth. EXC-4 and human CLICs have conserved functions in tubulogenesis, and CLICs and G-Rho/Rac signaling regulate tubulogenesis during blood vessel development. Therefore, our work defines a primordial role for EXC-4/CLICs in G-Rho/Rac-signaling during tubulogenesis. One Sentence SummaryG and Rho/Rac signaling regulates EXC-4/CLIC-mediated cell outgrowth during tubulogenesis in C. elegans, linking elements of G-protein signaling to the enigmatic CLIC family of proteins.

Norepinephrine activates β1-adrenergic receptors localized to the inner nuclear membrane in cortical astrocytes

Benton, K. C. — 2021-06-24

Studies in cardiomyocytes have established that adrenergic receptors, conventionally thought to initiate signaling events exclusively from the plasma membrane, can also localize to and signal from the nuclear membrane. Activation of these receptors by their endogenous cationic ligands requires transmembrane uptake mediated by organic cation transporter 3 (OCT3). We have demonstrated that OCT3 is densely localized to outer nuclear membranes in neurons and astrocytes, suggesting that nuclear adrenergic signaling is also present in the central nervous system. In this study, we examined the subcellular localization of {beta}1-adrenergic receptors, their G-protein signaling partners, and catecholamine transporters in mouse astrocytes. We identified a population of {beta}1-adrenergic receptors localized to astrocyte inner nuclear membranes. We demonstrated that key components of Gs-mediated signaling are localized to the nuclear compartment and identified OCT3 and other catecholamine transporters localized to plasma and nuclear membranes. Treatment of astrocytes with norepinephrine induced rapid increases in nuclear PKA activity which were blocked by pretreatment with inhibitors of catecholamine transport. These data indicate that nuclear {beta}1-adrenergic receptors are functionally coupled to Gs-coupled signaling mediators and that their activation by norepinephrine requires transporter-mediated uptake. These receptors represent a powerful mechanism by which norepinephrine may alter astrocyte gene expression and brain function.

AP-4 regulates neuronal lysosome composition, function and transport via regulating export of critical lysosome receptor proteins at the trans-Golgi network

Majumder, P. — 2021-06-25

The adaptor protein complex-4 or AP-4 is known to mediate autophagosome maturation through regulating sorting of transmembrane cargo such as ATG9A at the Golgi. There is a need to understand AP-4 function in neurons, as mutations in any of its four subunits cause a complex form of hereditary spastic paraplegia (HSP) with intellectual disability. While AP-4 has been implicated in regulating trafficking and distribution of cargo such as ATG9A and APP, little is known about its effect on neuronal lysosomal protein traffic, lysosome biogenesis and function. In this study, we demonstrate that in human iPSC-derived neurons AP-4 regulates lysosome composition, function and transport via regulating export of critical lysosomal receptors, including Sortilin 1, from the trans-Golgi network to endo-lysosomes. Additionally, loss of AP-4 causes endo-lysosomes to stall and build up in axonal swellings potentially through reduced recruitment of retrograde transport machinery to the organelle. These findings of axonal lysosome build-up are highly reminiscent of those observed in Alzheimers disease as well as in neurons modelling the most common form of HSP, caused by spastin mutations. Our findings implicate AP-4 as a critical regulator of neuronal lysosome biogenesis and altered lysosome function and axonal endo-lysosome transport as an underlying defect in AP-4 deficient HSP.

FRET and LRET Biosensors for Cell-based Imaging and Screening of Rac1 Activation

Pham, H. — 2021-06-26

Rac1 is a key regulator of several cell signaling pathways and dysregulated Rac1 activation has been implicated in cancer. Genetically encoded Forster resonance energy transfer (FRET) biosensors with enhanced dynamic range enabled live cell fluorescence imaging of Rac1 activity and a cell lysate-based assay of Rac1 inhibition in 96-well plates. We prepared HEK293 cell lines that stably expressed polypeptides with a general domain sequence (N- to C-terminus) of 1) FRET acceptor; 2) Rac/Cdc42 binding domain of human p21 protein kinase A (residues 68-150); 3) a linker domain; 4) FRET donor; and 5) full-length Rac1. Activated Rac1 binds to the protein kinase A domain, bringing donors and acceptors close together to increase FRET. We evaluated the effects on FRET signal dynamic range of alpha helical linkers comprised of alternating repeats of roughly four glutamate and four arginine or lysine residues. So-called ER/K linkers had limited effects on conventional FRET biosensors that incorporated the fluorescent protein (FP) pairs mCerulean/YPet, or mTFP1(cp227)/mVenus(cp229). Fluorometric measurements of cells that co-expresssed biosensors with positive (TIAM1) or negative (RhoGDI) Rac1 regulators revealed significant dynamic range enhancement in only one FP construct (mCerulean/YPet with 20 nm ER/K linker) relative to an analogous structure that incorporated an unstructured linker. We transfected this construct into a cell line that stably expressed a rapamycin-inducible c-Src analog (RapR-Src) and observed activation of Rac1 at protruding edges following rapamycin stimulation. In cells that expressed lanthanide-based FRET (LRET biosensors) that incorporated a luminescent terbium complex donor and GFP fluorescent acceptor, time-gated luminescence (TGL) measurements showed substantial gains in dynamic range that increased with linker length (up to 1200%). We robustly detected small molecule Rac1 inhibition following lysis of LRET biosensor-expressing cells grown directly in 96-well plates. The results herein highlight the potential of FRET and LRET biosensors with ER/K linkers for cell-based imaging and screening of protein activities.

Identification of novel translated small ORFs in Escherichia coli using complementary ribosome profiling approaches

Stringer, A. M. — 2021-07-03

Small proteins of <51 amino acids are abundant across all domains of life but are often overlooked because their small size makes them difficult to predict computationally, and they are refractory to standard proteomic approaches. Ribosome profiling has been used to infer the existence of small proteins by detecting the translation of the corresponding open reading frames (ORFs). Detection of translated short ORFs by ribosome profiling can be improved by treating cells with drugs that stall ribosomes at specific codons. Here, we combine the analysis of ribosome profiling data for Escherichia coli cells treated with antibiotics that stall ribosomes at either start or stop codons. Thus, we identify ribosome-occupied start and stop codons for ~400 novel putative ORFs with high sensitivity. The newly discovered ORFs are mostly short, with 365 encoding proteins of <51 amino acids. We validate translation of several selected short ORFs, and show that many likely encode unstable proteins. Moreover, we present evidence that most of the newly identified short ORFs are not under purifying selection, suggesting they do not impact cell fitness, although a small subset have the hallmarks of functional ORFs. IMPORTANCESmall proteins of <51 amino acids are abundant across all domains of life but are often overlooked because their small size makes them difficult to predict computationally, and they are refractory to standard proteomic approaches. Recent studies have discovered small proteins by mapping the location of translating ribosomes on RNA using a technique known as ribosome profiling. Discovery of translated sORFs using ribosome profiling can be improved by treating cells with drugs that trap initiating ribosomes. Here, we show that combining these data with equivalent data for cells treated with a drug that stalls terminating ribosomes facilitates the discovery of small proteins. We use this approach to discover 365 putative genes that encode small proteins in Escherichia coli.

Aberrant enteric neuromuscular system and dysbiosis in amyotrophic lateral sclerosis

Zhang, Y.-g. — 2021-07-14

BackgroundEmerging evidence has demonstrated that microbiota directly affects the enteric neuron system (ENS) and smooth muscle cell functions via metabolic products or endogenous bacterial components. Amyotrophic Lateral Sclerosis is a neuromuscular disease characterized by the progressive death of motor neurons and muscle atrophy. The GI symptoms in patients were largely ignored or underestimated, especially before the diagnosis of ALS. The relationship between enteric neuromuscular system and microbiome in ALS progression is unknown. MethodsWe performed longitudinal studies on the ENS and microbiome in the ALS human-SOD1G93A transgenic G93A mice. We treated age-matched wild-type and ALS mice with bacterial product butyrate or antibiotics to investigate microbiome and neuromuscular functions. Intestinal motility, microbiome, an ENS marker GFAP, a smooth muscle marker (SMMHC), and human colonoids have been examined. The distribution of human-G93A-SOD1 (Superoxide Dismutase 1) protein was tested as an indicator of ALS progression. ResultsAt 2-month-old before ALS onset, G93A mice had significant lower intestinal motility, decreased grip strength, and reduced time in the rotarod. We observed increased GFAP and decreased SMMHC expression. These changes correlated with consistent increased aggregation of mutated SOD1G93A in the colon, small intestine, and spinal cord. Butyrate and antibiotic treatment showed a significantly longer latency to fall in the rotarod test, reduced SOD1G93A aggregation, and enhanced ENS and muscle function. Feces from 2-month-old SOD1G93A mice significantly enhanced SOD1G93A aggregation in human colonoids transfected with a SOD1G93A-GFP plasmid. Longitudinal studies of microbiome data further showed the altered bacterial community related with autoimmunity (e.g., Clostridium sp. ASF502, Lachnospiraceae bacterium A4), inflammation (e.g., Enterohabdus Muris,), and metabolism (e.g., Desulfovibrio fairfieldensis) at 1- and 2-month-old SOD1G93A mice, suggesting the early microbial contribution to the pathological changes. ConclusionsWe have demonstrated a novel link between microbiome, hSOD1G93A aggregation, and intestinal mobility. Dysbiosis occurred at the early stage of the ALS mice before observed mutated-SOD1 aggregation, slow intestinal motility, and dysfunction of ENS. Manipulating the microbiome improves the muscle performance of SOD1G93A mice. Our study provides insights into fundamentals of intestinal neuromuscular structure/function and microbiome in ALS.

Binding Free energy Decomposition and Multiple Unbinding Paths of Buried Ligands in a PreQ1 Riboswitch

Hu, G. — 2021-07-14

Riboswitches are naturally occurring RNA elements that control bacterial gene expression by binding to specific small molecules. They serve as important models for RNA-small molecule recognition and have also become a novel class of targets for developing antibiotics. Here, we carried out conventional and enhanced-sampling molecular dynamics (MD) simulations, totaling 141.5 s, to characterize the determinants of binding free energies and unbinding paths for the cognate and synthetic ligands of a PreQ1 riboswitch. Binding free energy analysis showed that two triplets of nucleotides U6-C15-A29 and G5-G11-C16, contribute the most to the binding of the cognate ligands, by hydrogen bonding and by base stacking, respectively. Mg2+ ions are essential in stabilizing the binding pocket. For the synthetic ligands, the hydrogen-bonding contributions of the U6-C15-A29 triplet are significantly compromised, and the bound state resembles the apo state in several respects, including the disengagement of the C15-A14-A13 and A32-G33 base stacks. The bulkier synthetic ligands lead to significantly loosening of the binding pocket, including extrusion of the C15 nucleobase and a widening of the C15-C30 groove. Enhanced-sampling simulations further revealed that the cognate and synthetic ligands unbind in almost opposite directions. Our work offers new insight for designing riboswitch ligands.

Epigenetic modulation of selected immune response genes and altered functions of T lymphocytes and macrophages collectively contribute to autoimmune diabetes protection

Jayaraman, S. — 2021-07-16

We have previously demonstrated that treatment of female NOD mice with the histone deacetylase inhibitor Trichostatin A (TSA) bestowed irreversible protection against autoimmune diabetes. Herein we show that drug treatment diminished the infiltration of the pancreas with CD4+ and CD8+ T cells and Ly-6C+ monocytes. Significantly, TSA administration selectively repressed the expression of a set of genes exaggerated during diabetes and constitutively expressed primarily in the spleen and rarely in the pancreas. These genes encode lymphokines, macrophage-associated determinants, and transcription factors. Although the copy numbers of many histone deacetylases increased during diabetes in the spleen and pancreas, only those upregulated in the spleen were rendered sensitive to repression by TSA treatment. The T lymphocytes derived from drug-treated donors displayed diminished diabetogenic potential following transfer into immunodeficient NOD.scid mice. In the immunocompromised recipients, diabetes caused by the transfer of activated T lymphocytes from untreated diabetic mice was hampered by the co-transfer of highly purified splenic Ly-6C+ macrophages from drug-treated mice. However, the transfer of Ly-6C+ macrophages from drug-treated mice failed to block ongoing diabetes in wild-type NOD mice. These data demonstrate that the modified gene expression and functional alteration of T lymphocytes and macrophages collectively contribute to diabetes protection afforded by the histone modifier in female NOD mice.

Gestational insulin resistance is mediated by the gut microbiome-indoleamine 2,3-dioxygenase axis

Priyadarshini, M. — 2021-07-22

Background and aimsNormal gestation involves reprogramming of maternal gut microbiome (GM) that may contribute to maternal metabolic changes by unclear mechanisms. This study aimed to understand the mechanistic underpinnings of GM - maternal metabolism interaction. MethodsThe GM and plasma metabolome of CD1, NIH-Swiss and C57BL/6J mice were analyzed using 16S rRNA sequencing and untargeted LC-MS throughout gestation and postpartum. Pharmacologic and genetic knockout mouse models were used to identify the role of indoleamine 2,3-dioxygenase (IDO1) in pregnancy-associated insulin resistance (IR). Involvement of gestational GM in the process was studied using fecal microbial transplants (FMT). ResultsSignificant variation in gut microbial alpha diversity occurred throughout pregnancy. Enrichment in gut bacterial taxa was mouse strain and pregnancy time-point specific, with species enriched at gestation day 15/19 (G15/19), a point of heightened IR, distinct from those enriched pre- or post- pregnancy. Untargeted and targeted metabolomics revealed elevated plasma kynurenine at G15/19 in all three mouse strains. IDO1, the rate limiting enzyme for kynurenine production, had increased intestinal expression at G15, which was associated with mild systemic and gut inflammation. Pharmacologic and genetic inhibition of IDO1 inhibited kynurenine levels and reversed pregnancy-associated IR. FMT revealed that IDO1 induction and local kynurenine levels effects on IR derive from the GM in both mouse and human pregnancy. ConclusionsGM changes accompanying pregnancy shift IDO1-dependent tryptophan metabolism toward kynurenine production, intestinal inflammation and gestational IR, a phenotype reversed by genetic deletion or inhibition of IDO1.

Epigenetic reprogramming ameliorates type 1 diabetes by decreasing the generation of Th1 and Th17 subsets and restoring self-tolerance in CD4+ T cells

Patel, V. — 2021-07-25

The histone modifier Trichostatin A (TSA) ameliorated diabetes and repressed IFN-{gamma} and IL-17A expression in prediabetic female NOD mice. Purified CD4+ cells could be polarized ex vivo into Th1 and Th17 subsets, which comparably transferred diabetes into NOD.scid mice. Polarized Th1 cells were devoid of IL-17A-producing cells and did not transdifferentiate into Th17 cells in an immunodeficient environment. However, Th17 cells had contaminant Th1 cells, which expressed IFN-{gamma} upon adoptive transfer into lymphopenic recipients. Notably, TSA treatment abrogated the transfer of diabetes by CD4+ T-cells cultured under Th1 or Th17 polarizing conditions accompanied by the absence of Ifng and Il17a expression in NOD.scid recipients. Significantly, the histone modifier restored the ability of CD4+ but not CD8+ T-cells to undergo CD3-mediated apoptosis ex vivo in a caspase-dependent manner. Thus, the histone modifier afforded protection against autoimmune diabetes by negative regulation of signature lymphokines and restitution of self-tolerance in CD4+ T cells.

The kpc-1 (furin) 3'UTR promotes dendritic transport and local translation of mRNAs to regulate dendrite branching and self-avoidance of a nociceptive neuron

Zou, Y. — 2021-08-05

A recently reported Schizophrenia-associated genetic variant in the 3UTR of the human furin gene, a homolog of C. elegans kpc-1, highlights an important role of the furin 3UTR in neuronal development(1). We isolate three kpc-1 mutants that display abnormal dendrite arborization in PVD neurons and defective male mating behaviors. We show that the kpc-1 3UTR participates in dendrite branching and self-avoidance. The kpc-1 3UTR facilitates mRNA localization to branching points and contact points between sibling dendrites and promotes local protein synthesis. We identify a secondary structural motif in the kpc-1 3UTR required for dendrite self-avoidance. Animals with dma-1 receptor over-expression exhibit similar dendrite branching and self-avoidance defects that are suppressed with kpc-1 over-expression. Our results support a model in which KPC-1 proteins are synthesized at branching points and contact points to locally down-regulate DMA-1 receptors to promote dendrite branching and self-avoidance of a mechanosensory neuron important for male courtship.

Preferential interactions of a crowder protein with the specific binding site of a native protein complex

Dong, X. — 2021-08-09

The crowded cellular environments provide ample opportunities for proteins to interact with bystander macromolecules, yet direct evidence, let alone residue-specific information, for such nonspecific binding is rare. Here, by combining NMR spectroscopy and atomistic modeling, we investigated how crowders influence the association equilibrium and kinetics of two protein partners, EIN and HPr. Ficoll-70 increases the EIN-HPr binding affinity whereas bovine serum albumin (BSA) decreases the affinity. The opposite effects of the two crowders are quantitatively explained by atomistic modeling, which shows that the stabilizing effect of Ficoll-70 arises from volume exclusion favoring the bound state. In contrast, the destabilizing effect of BSA arises from preferential soft interactions with the free state; notably, BSA has favorable electrostatic interactions with positively charged HPr residues within the EIN-binding site. Some of the residues from this site indeed experience significant chemical shift perturbation when titrated with BSA, while the relaxation rates of HPr backbone amides exhibit overall elevation. Furthermore, relaxation dispersion data indicate that Ficoll-70 and BSA both slow down the EIN-HPr association rate, but change the dissociate rate in opposite directions. The observations on kinetics are accounted for by two effects of the crowders: increasing the solution microviscosity and reshaping the EIN-HPr interaction energy surface. The kind of preferential interactions between BSA and HPr that leads to competition with EIN should be prevalent in cellular environments. Our NMR results and atomistic modeling provide benchmarks, at both qualitative and quantitative levels, for the effects of crowded cellular environments on protein-protein specific interactions. Significance StatementAlthough nonspecific binding of crowder macromolecules with functional proteins is likely prevalent in vivo, direct evidence is rare. Here we present NMR characterizations showing that bovine serum albumin preferentially interacts with a specific binding site on HPr, leading to competition with the latters partner EIN. The preferential interactions result in destabilization of the EIN-HPr native complex and speedup of its dissociation, contrary to expectations from excluded-volume and viscosity effects. Atomistic modeling of macromolecular crowding rationalizes the experimental observations, and provides qualitative and quantitative insight into the influences of the crowded cellular environment on protein-protein specific interactions. Our work also has implications for evolution, regarding how nonspecific binding can be either minimized or exploited for gaining new functions.

Minimalistic 3D Chromatin Models: Sparse Interactions in Single Cells Drive the Chromatin Fold and Form Many-Body Units

Liang, J. — 2021-08-09

Computational modeling of 3D chromatin plays an important role in understanding the principles of genome organization. We discuss methods for modeling 3D chromatin structures, with focus on a minimalistic polymer model which inverts population Hi-C into high-resolution, high-coverage single-cell chromatin conformations. Utilizing only basic physical properties such as nuclear volume and no adjustable parameters, this model uncovers a few specific Hi-C interactions (15-35 for enhancerrich loci in human cells) that can fold chromatin into individual conformations consistent with single-cell imaging, Dip-C, and FISH-measured genomic distance distributions. Aggregating an ensemble of conformations also reproduces population Hi-C interaction frequencies. Furthermore, this single-cell modeling approach allows quantification of structural heterogeneity and discovery of specific many-body units of chromatin interactions. This minimalistic 3D chromatin polymer model has revealed a number of insights: 1) chromatin scaling rules are a result of volume-confined polymers; 2) TADs form as a byproduct of 3D chromatin folding driven by specific interactions; 3) chromatin folding at many loci is driven by a small number of specific interactions; 4) cell subpopulations equipped with different chromatin structural scaffolds are developmental stage-dependent; and 5) characterization of the functional landscape and epigenetic marks of many-body units which are simultaneously spatially co-interacting within enhancer-rich, euchromatic regions. The implications of these findings in understanding the genome structure-function relationship are also discussed.

DDX41 is needed for pre-and post-natal hematopoietic stem cell differentiation in mice

Ma, J. — 2021-08-13

DDX41 is a tumor suppressor frequently mutated in human myeloid neoplasms. DDX41 binds to DNA/RNA hybrids and interacts with spliceosome component (1, 2). How it affects hematopoiesis is still unclear. Using a knockout mouse model, we demonstrate that DDX41 is required for mouse hematopoietic stem and progenitor cell (HSPC) survival and differentiation. Lack of DDX41 particularly affected myeloid progenitor development, starting at embryonic day 13.5. DDX41-deficient fetal liver and adult bone marrow (BM) cells were unable to rescue mice from lethal irradiation after transplantation. DDX41 knockout stem cells were also defective in ex vivo colony forming assays. RNASeq analysis of lineage-negative, cKit+Sca1+ cells isolated from fetal liver demonstrated that the expression of many genes associated with hematopoietic differentiation were altered in DDX41 knockout cells. Furthermore, altered splicing of genes involved in key biological processes were observed. Our data reveal a critical role for DDX41 in HSPC differentiation and myeloid progenitor development, likely through its regulation of gene expression programs and splicing. SignificanceDDX41 is a tumor suppressor in hematologic malignancies. However, whether DDX41 functions in hematopoiesis and myeloid cell differentiation is not known. Here we show that in mice, loss of DDX41 in hematopoietic stem cells (HSCs) leads to defects in hematopoietic development. The myeloid lineage was particularly affected as early as pre-natal stages. Transcriptional profiling of embryonic HSCs revealed that there were global changes in gene expression and splicing due to lack of DDX41. Collectively, the study uncovers a new function of DDX41 in HSC differentiation and could provide molecular targets for treatment of myeloid differentiation disorders.

Desolvation Energy Explains Partitioning of Client Proteins into Condensates

Villegas, J. — 2021-08-17

Membraneless organelles are cellular compartments that form by liquid-liquid phase separation of one or more components. Other molecules, such as other proteins and nucleic acids, will distribute between the cytoplasm and the liquid compartment in accordance with the thermodynamic drive to lower the free energy of the system. The resulting distribution colocalizes molecular species, to carry out a diversity of functions. Two factors could drive this partitioning: the difference in solvation between the dilute versus dense phase, and intermolecular interactions between the client and scaffold proteins. Here, we develop a set of knowledge-based potentials that allow for the direct comparison between desolvation energy and pairwise interaction energy terms, and use these to examine experimental data from two systems: protein cargo dissolving within phase-separated droplets made from FG repeat proteins of the nuclear pore complex, and client proteins dissolving within phase-separated FUS droplets. We find close agreement between desolvation energies of the client proteins and the experimentally determined values of the partition coefficients, while pairwise interaction energies between client and scaffold show weaker correlations. These results show that client stickiness is sufficient to explain differential partitioning of clients within these two phase-separated systems without taking into account the composition of the condensate. This suggests that selective trafficking of client proteins to distinct membraneless organelles requires recognition elements beyond the client sequence composition.

CREB5 reprograms nuclear interactions to promote resistance to androgen receptor targeting therapies

Hwang, J. H. — 2021-08-18

Metastatic castration resistant prostate cancers (mCRPC) are treated with therapies that antagonize the androgen receptor (AR). Nearly all patients develop resistance to AR-targeted therapies (ART). Our previous work identified CREB5 as an upregulated target gene in human mCRPC that promoted resistance to all clinically-approved ART. The mechanisms by which CREB5 promotes progression of mCRPC or other cancers remains elusive. Integrating ChIP-seq and rapid immunoprecipitation and mass spectroscopy of endogenous proteins (RIME), we report that cells overexpressing CREB5 demonstrate extensive reprogramming of nuclear protein-protein interactions in response to the ART agent enzalutamide. Specifically, CREB5 physically interacts with AR, the pioneering actor FOXA1, and other known co-factors of AR and FOXA1 at transcription regulatory elements recently found to be active in mCRPC patients. We identified a subset of CREB5/FOXA1 co-interacting nuclear factors that have critical functions for AR transcription (GRHL2, HOXB13) while others (TBX3, NFIC) regulated cell viability and ART resistance and were amplified or overexpressed in mCRPC. Upon examining the nuclear protein interactions and the impact of CREB5 expression on the mCRPC patient transcriptome, we found CREB5 was associated with TGF{beta} and Wnt signaling and epithelial to mesenchymal transitions, implicating these pathways in ART resistance. Overall, these observations define the molecular interactions among CREB5, FOXA1, and pathways that promote ART resistance.

Repair of APOBEC3G-mutated retroviral DNA in vivo is facilitated by the host enzyme uracil DNA glycosylase 2

Salas-Briceno, K. — 2021-08-25

Apolipoprotein B mRNA Editing Enzyme Catalytic Subunit 3 (APOBEC3) proteins are critical for the control of infection by retroviruses. These proteins deaminate cytidines in negative strand DNA during reverse transcription, leading to G to A changes in coding strands. Uracil DNA glycosylase (UNG) is a host enzyme that excises uracils in genomic DNA, which the base excision repair machinery then repairs. Whether UNG removes uracils found in retroviral DNA after APOBEC3-mediated mutation is not clear, and whether this occurs in vivo has not been demonstrated. To determine if UNG plays a role in the repair of retroviral DNA, we used APOBEC3G (A3G) transgenic mice which we showed previously had extensive deamination of murine leukemia virus (MLV) proviruses. The A3G transgene was crossed onto an UNG and mouse APOBEC3 knockout background (UNG-/-APO-/-) and the mice were infected with MLV. We found that virus infection levels were decreased in A3G UNG-/-APO-/- compared to A3G APO-/- mice. Deep sequencing of the proviruses showed that there were significantly higher levels of G-to-A mutations in proviral DNA from A3G transgenic UNG-/-APO-/- than A3G transgenic APO-/- mice, suggesting that UNG plays a role in the repair of uracil-containing proviruses. In in vitro studies, we found that cytoplasmic viral DNA deaminated by APOBEC3G was uracilated. In the absence of UNG, the uracil-containing proviruses integrated at higher levels into the genome than did those made in the presence of UNG. Thus, UNG also functions in the nucleus prior to integration by nicking uracil-containing viral DNA, thereby blocking integration. These data show that UNG plays a critical role in the repair of the damage inflicted by APOBEC3 deamination of reverse-transcribed DNA. ImportanceWhile APOBEC3-mediated mutation of retroviruses is well-established, what role the host base excision repair enzymes play in correcting these mutations is not clear. This question is especially difficult to address in vivo. Here, we use a transgenic mouse developed by our lab that expresses human APOBEC3G and also lacks the endogenous uracil DNA glycosylase (Ung) gene, and show that UNG removes uracils introduced by this cytidine deaminase in MLV reverse transcripts, thereby reducing G-to-A mutations in proviruses. Furthermore, our data suggest that UNG removes uracils at two stages in infection - in unintegrated nuclear viral reverse transcribed DNA, resulting in its degradation and second, in integrated proviruses, resulting in their repair. These data suggest that retroviruses damaged by host cytidine deaminases take advantage of the host DNA repair system to overcome this damage.

Relationship between bacterial phylotype and specialized metabolite production in the culturable microbiome of two freshwater sponges

Clark, C. M. — 2021-08-26

Microbial drug discovery programs rely heavily on accessing bacterial diversity from the environment to acquire new specialized metabolite (SM) lead compounds for the therapeutic pipeline. Therefore, knowledge of how certain bacterial taxa are distributed in nature, in addition to the degree of variation of SM production within those taxa, is critical to informing these front-end discovery efforts and making the overall sample collection and bacterial library creation process more efficient. In the current study we employed MALDI-TOF mass spectrometry and the bioinformatics pipeline IDBac to analyze diversity within phylotype groupings and SM profiles of hundreds of bacterial isolates from two Eunapius fragilis freshwater sponges, collected 1.5 km apart. We demonstrated that within two sponge samples of the same species, the culturable bacterial populations contained significant overlap in approximate genus-level phylotypes but mostly non-overlapping populations of isolates when grouped lower than the level of genus. Further, correlations between bacterial phylotype and SM production varied at the species level and below, suggesting SM distribution within bacterial taxa must be analyzed on a case-by-case basis. Our results suggest that two E. fragilis freshwater sponges collected in similar environments can exhibit large culturable diversity on a species-level scale, thus researchers should scrutinize the isolates with analyses that take both phylogeny and SM production into account in order to optimize the chemical space entering into a downstream bacterial library.

Retinoschisin deficiency induces persistent aberrant waves of activity affecting neuroglial signaling in the retina

Eleftheriou, C. G. — 2021-08-28

Genetic disorders which present during development make treatment strategies particularly challenging because there is a need to disentangle primary pathophysiology from downstream dysfunction caused at key developmental stages. To provide a deeper insight into this question, we studied a mouse model of X-linked juvenile retinoschisis (XLRS), an early onset inherited condition caused by mutations in the RS1 gene encoding retinoschisin (RS1) and characterized by cystic retinal lesions and early visual deficits. Using an unbiased approach in expressing the fast intracellular calcium indicator GCaMP6f in neuronal, glial, and vascular cells of the retina of mice lacking RS1, we found that initial cyst formation is paralleled by the appearance of aberrant spontaneous neuro-glial signals as early as postnatal day 13. These presented as glutamate-driven wavelets of neuronal activity and sporadic radial bursts of activity by Muller glia, spanning all retinal layers and disrupting light-induced signaling. This study highlights a critical role for RS1 in early retinal development with a potential to disrupt circuit formation to central targets. Additionally, it confers a functional role to RS1 beyond the scope of an adhesion molecule and identifies an early onset for dysfunction, a potential temporal target for therapeutic intervention and diagnosis. Significance Statement/ HighlightsO_LIPhotoreceptor inner segments express Rs1 at P5, after which RS1 protein is detected in the inner segments by P9 and throughout the retina at later ages, with structural abnormalities observed by optical coherence tomography at P13 in Rs1 mutant mouse models. C_LIO_LIAberrant glutamate-driven wavelets identified by GCaMP6f-based analyses are a novel pathophysiological feature of RS1 deficient mice that emerge after maximal RS1 expression. C_LIO_LIMuller glia display abnormal radial glutamate-driven coordinated and sporadic bursts of activity in RS1-deficient mice. C_LIO_LIThese data identify a novel pathophysiological feature of RS1-deficient mice and define a window where treatments might be most effective. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/457777v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@3f27eborg.highwire.dtl.DTLVardef@55e5a4org.highwire.dtl.DTLVardef@1577435org.highwire.dtl.DTLVardef@8e55b3_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mapping the Energy Landscape of PROTAC-Mediated Protein-protein Interactions

Villegas, J. A. — 2021-09-01

One of the principal difficulties in computational modeling of macromolecules is the vast conformational space that arises out of large numbers of atomic degrees of freedom. This problem is a familiar issue in the area of protein-protein docking, where models of protein complexes are generated from the monomeric subunits. Although restriction of molecular flexibility is a commonly used approximation that decreases the dimensionality of the problem, the seemingly endless number of possible ways two binding partners can interact generally necessitates the use of further approximations to explore the search space. Recently, growing interest in using computational tools to build predictive models of PROTAC-mediated complexes has led to the application of state-of-the-art protein-protein docking techniques to tackle this problem. Additionally, the atomic degrees of freedom introduced by flexibility of linkers used in the construction of PROTACs further expands the configurational search space, a problem that can be tackled with conformational sampling tools. However, repurposing existing tools to carry out protein-protein docking and linker conformer generation independently results in extensive sampling of structures incompatible with PROTAC-mediated complex formation. Here we show that it is possible to restrict the search to the space of protein-protein conformations that can be bridged by a PROTAC molecule with a given linker composition by using a cyclic coordinate descent algorithm to position PROTACs into complex-bound configurations. We use this methodology to construct a picture of the energy landscape of PROTAC-mediated interactions in a model test case, and show that the global minimum lies in the space of native-like conformations.

Redox-mediated inactivation of the transcriptional repressor C3600 makes uropathogenic Escherichia coli exquisitely resistant to reactive chlorine species

Sultana, S. — 2021-09-01

The ability to overcome stressful environments is critical for pathogen survival in the host. One challenge for bacteria is the exposure to reactive chlorine species (RCS), which are generated by innate immune cells as critical part of the oxidative burst. Hypochlorous acid (HOCl) is the most potent antimicrobial RCS and associated with extensive macromolecular damage in the phagocytized pathogen. However, bacteria have evolved defense strategies to alleviate the effects of HOCl-mediated damage. Among these are RCS-sensing transcriptional regulators that control the expression of HOCl-protective genes under non- and HOCl stress. Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils during pathogenesis, however, their responses to and defenses of HOCl are still completely unexplored. Here, we present evidence that UPEC strains tolerate higher levels of HOCl and are better protected from neutrophil-mediated killing compared to other E. coli. Transcriptomic analysis of HOCl-stressed UPEC revealed the upregulation of an operon consisting of three genes, one of which encodes the transcriptional regulator C3600. We identified C3600 as a HOCl-sensing transcriptional repressor, which, under non-stress conditions, is bound to the operator and represses the expression of its target genes. During HOCl exposure, however, the repressor forms reversible intermolecular disulfide bonds and dissociates from the DNA resulting in the de-repression of the operon. Deletion of one of the target genes renders UPEC significantly more susceptible to HOCl indicating that the HOCl-mediated induction of the regulon plays a major role for UPECs HOCl resistance. IMPORTANCEHow do pathogens deal with antimicrobial oxidants produced by the innate immune system during infection? Uropathogenic Escherichia coli (UPEC), the most common etiological agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils and, therefore, must counter elevated levels of the antimicrobial oxidant HOCl to establish infection. Our study provides fundamentally new insights into a defense mechanism that enables UPEC to fend off the toxic effects of HOCl stress. Intriguingly, the defense system is predominantly found in UPEC and absent in non-invasive enteropathogenic E. coli. Our data suggest that expression of the target gene c3601 is exclusively responsible for UPECs increased HOCl tolerance in culture and therefore potentially contributes to UPECs survival during phagocytosis. Thus, this novel HOCl stress defense system could potentially serve as an attractive drug target to increase the bodys own capacity to fight UTIs.

Interrogation of the integrated mobile genetic elements in gut-associated Bacteroidaceae with a consensus prediction approach

Campbell, D. E. — 2021-09-04

Exploration of mobile genetic element (MGE) diversity and relatedness is vital to understanding microbial communities, especially the gut microbiome, where the mobilization of antibiotic resistance and pathogenicity genes has important clinical consequences. Current MGE prediction tools are biased toward elements similar to previously-identified MGEs, especially tailed phages of proteobacterial hosts. Further, there is a need for methods to examine relatedness and gene sharing among MGEs. We present VICSIN, a consensus approach for MGE prediction and clustering of predictions to provide classification. Testing of VICSIN on datasets of Pseudomonas aeruginosa and Bacteroides fragilis genomes suggests VICSIN is the optimal approach to predict integrated MGEs from poorly-explored host taxa, because of its increased sensitivity and accuracy. We applied VICSIN to a dataset of gut-associated Bacteroidaceae genomes, identifying 816 integrated MGEs falling into 95 clusters, most of which are novel. VICSINs fast and simple network-building scheme revealed a high degree of gene sharing within and between related MGE clusters. Shared gene functions across MGEs include core mobilization functions and accessory gene content, such as type VI secretion systems and antibiotic resistance genes. The MGEs identified here encode a large portion of unknown gene content, emphasizing the fact that the full diversity of MGEs and the factors they encode remain very poorly understood. Together, this work motivates more exploration of the gut mobilome, which is likely one of the most potent drivers of microbial evolution in the human microbiome. IMPORTANCEMobile genetic elements (MGEs), including phages and integrative and conjugative elements (ICEs), drive the diversity and function of microbial communities through horizontal gene transfer. Current tools to predict MGEs in genomic sequence data are highly focused on phages, and are biased against the discovery of novel MGEs. We present VICSIN, a consensus approach to MGE prediction that is able to find a diversity of MGEs, particularly in poorly-understood bacterial taxa. By applying VICSIN to a large database of diverse Bacteroidaceae genomes, we have been able to get a distinct view of the gut mobilome, extending beyond the phageome. These novel MGEs belong to related groups, sharing a significant amount of functional gene content within and between groups, supporting a mosaic model of evolution for ICEs. Understanding how phages evolve in Bacteroidaceae hosts, however, remains elusive and highlights the need for more experimental research.

Cyclic mechanical stresses alter erythrocyte membrane composition and microstructure and trigger macrophage phagocytosis

Garcia-Herreros, A. — 2021-09-11

Red blood cells (RBCs) are cleared from the circulation when they become damaged or display aging signals targeted by macrophages. This process occurs mainly in the spleen, where blood flows through submicrometric constrictions called inter-endothelial slits (IES), subjecting RBCs to large-amplitude deformations. In this work, we circulated RBCs through microfluidic devices containing microchannels that replicate the IES. The cyclic mechanical stresses experienced by the cells affected their biophysical properties and molecular composition, accelerating cell aging. Specifically, RBCs quickly transitioned to a more spherical, less deformable phenotype that hindered microchannel passage, causing hemolysis. This transition was associated with the release of membrane vesicles, which self-extinguished as the spacing between membrane-cytoskeleton linkers became tighter. Proteomics analysis of the mechanically aged RBCs revealed significant losses of essential proteins involved in antioxidant protection, gas transport, and cell metabolism. Finally, we show that these changes made mechanically aged RBCs more susceptible to macrophage phagocytosis. These results provide a comprehensive model to explain how physical stress induces RBC clearance in the spleen.

Dense cortical input to the rostromedial tegmental nucleus mediates aversive signaling

Glover, E. J. — 2021-09-11

AO_SCPLOWBSTRACTC_SCPLOWThe rostromedial tegmental nucleus (RMTg) encodes negative reward prediction error (RPE) and plays an important role in guiding behavioral responding to aversive stimuli. While initial studies describing the RMTg revealed the presence of cortical afferents, the density and distribution of this input has not been explored in detail. In addition, the functional consequences of cortical modulation of RMTg signaling are only just beginning to be investigated. The current study anatomically and functionally characterizes cortical input to the RMTg in rats. Findings from this work reveal dense input spanning the entire medial prefrontal cortex (PFC) as well as the orbitofrontal cortex and anterior insular cortex. Afferents were most dense in the dorsomedial subregion of the PFC (dmPFC), an area which has also been implicated in both RPE signaling and aversive responding. RMTg-projecting dmPFC neurons originate in layer V and collateralize extensively throughout the brain. In-situ mRNA hybridization further revealed that neurons in this circuit are predominantly D1 receptor-expressing with a high degree of D2 receptor colocalization. Optogenetic stimulation of dmPFC terminals in the RMTg drives avoidance, and cFos expression is enhanced in this neural circuit during exposure to aversive stimuli. Exposure to such aversive stimuli results in significant physiological and structural plasticity suggestive of a loss of top-down modulation of RMTg-mediated signaling. Altogether, these data reveal the presence of a prominent cortico-subcortical projection involved in adaptive behavioral responding and provide a foundation for future work aimed at exploring alterations in circuit function in diseases characterized by deficits in cognitive control over the balance between reward and aversion.

Multiphase Organization Is a Second Phase Transition Within Multi-Component Biomolecular Condensates

Mazarakos, K. — 2021-09-16

We present a mean-field theory for the multiphase organization of multi-component biomolecular condensates and validate the theory by molecular dynamics simulations of model mixtures. A first phase transition results in the separation of the dense phase from the bulk phase. In a second phase transition, the components in the dense phase demix to localize in separate regions that attach to each other. The second phase transition occurs when the strength of cross-species attraction goes below the mean strength of the self-attraction of the individual species and reaches a critical value. At a given strength of cross-species attraction, both of the phase transitions can be observed by decreasing temperature, leading first to phase separation and then to demixing of the dense phase. The theory and simulations establish the disparity in strength between self and cross-species attraction as a main driver for the multiphase organization of multi-component biomolecular condensates. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/460104v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@e17fdcorg.highwire.dtl.DTLVardef@9168deorg.highwire.dtl.DTLVardef@7c07bcorg.highwire.dtl.DTLVardef@e0077d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Photoinduced isomerization sampling of retinal in bacteriorhodopsin

Ren, Z. — 2021-09-16

Photoisomerization of retinoids inside a confined protein pocket represents a critical chemical event in many important biological processes from animal vision, non-visual light effects, to bacterial light sensing and harvesting. Light driven proton pumping in bacteriorhodopsin entails exquisite electronic and conformational reconfigurations during its photocycle. However, it has been a major challenge to delineate transient molecular events preceding and following the photoisomerization of the retinal from noisy electron density maps when varying populations of intermediates coexist and evolve as a function of time. Here I report several distinct early photoproducts deconvoluted from the recently observed mixtures in time-resolved serial crystallography. This deconvolution substantially improves the quality of the electron density maps hence demonstrates that the all-trans retinal undergoes extensive isomerization sampling before it proceeds to the productive 13-cis configuration. Upon light absorption, the chromophore attempts to perform trans-to-cis isomerization at every double bond together with the stalled anti-to-syn rotations at multiple single bonds along its polyene chain. Such isomerization sampling pushes all seven transmembrane helices to bend outward, resulting in a transient expansion of the retinal binding pocket, and later, a contraction due to recoiling. These ultrafast responses observed at the atomic resolution support that the productive photoreaction in bacteriorhodopsin is initiated by light-induced charge separation in the prosthetic chromophore yet governed by stereoselectivity of its protein pocket. The method of a numerical resolution of concurrent events from mixed observations is also generally applicable. Significance StatementPhotoisomerization of retinal is a critical rearrangement reaction in many important biological processes from animal vision, non-visual light effects, to bacterial light sensing and harvesting. It has been a major challenge to visualize rapid molecular events preceding and following photoisomerization so that many protein functions depending on such reaction remain vaguely understood. Here I report a direct observation of the stereoselectivity of bacteriorhodopsin hence delineate the structural mechanism of isomerization. Upon a light-induced charge separation, the retinal in a flat conformation attempts to perform double bond isomerization and single bond rotation everywhere along its polyene chain before it proceeds to the specific, productive configuration. This observation improves our understanding on how a non-specific attraction force could drive a specific isomerization.

Amyloid β oligomer selective antibodies for Alzheimers therapeutics and diagnostics

Viola, K. L. — 2021-09-21

Improvements have been made in the diagnosis of Alzheimers disease (AD), manifesting mostly in the development of in vivo imaging methods that allow for the detection of pathological changes in AD by MRI and PET scans. Many of these imaging methods, however, use agents that probe amyloid fibrils and plaques - species that do not correlate well with disease progression and are not present at the earliest stages of the disease. Amyloid {beta} oligomers (A{beta}Os), rather, are now widely accepted as the A{beta} species most germane to AD onset and progression. Here we report evidence further supporting the role of A{beta}Os as pathological instigators of AD and introduce promising anti-A{beta}O diagnostic probes capable of distinguishing the 5xFAD mouse model from wild type mice by PET and MRI. In a developmental study, A{beta} oligomers in 5xFAD mice were found to appear at 3 months of age, just prior to the onset of memory dysfunction, and spread as memory worsened. The increase of A{beta}Os is prominent in the subiculum and correlates with concomitant development of reactive astrocytosis. The impact of these A{beta}Os on memory is in harmony with findings that intraventricular injection of synthetic A{beta}Os into wild type mice induced hippocampal dependent memory dysfunction within 24 hours. Compelling support for the conclusion that endogenous A{beta}Os cause memory loss was found in experiments showing that intranasal inoculation of A{beta}O-selective antibodies into 5xFAD mice completely restored memory function, measured 30-40 days post-inoculation. These antibodies, which were modified to give MRI and PET imaging probes, were able to distinguish 5xFAD mice from wild type littermates. These results provide strong support for the role of A{beta}Os in instigating memory loss and salient AD neuropathology, and they demonstrate that A{beta}O selective antibodies have potential both for therapeutics and for diagnostics.

Multi-modal profiling of the extracellular matrix of human fallopian tubes and serous tubal Intraepithelial carcinomas

Renner, C. — 2021-09-23

Recent evidence supports the fimbriae of the fallopian tube as a potential origin site for high-grade serous ovarian cancer (HGSOC). The progression of many solid tumors is accompanied by changes in the microenvironment, including alterations of the extracellular matrix (ECM). The ECM of fallopian tube and HGSOC has not been well characterized. Therefore, we sought to determine the ECM composition of the benign fallopian tube and how it changes with the onset of serous intraepithelial carcinomas (STICs), precursor of HGSOC. The ECM composition of benign human fallopian tube was first defined from a meta-analysis of published proteomic datasets and identified 190 ECM proteins. We then conducted de novo proteomics using ECM enrichment and identified 88 proteins, 7 of which were not identified in prior studies. We further investigated the levels and localization of seven of these ECM proteins (type I, III, and IV collagens, fibronectin, laminin, versican, perlecan) and hyaluronic acid using multi-spectral immunohistochemical staining of fimbriae from patients with benign conditions or STICs. Quantification revealed an increase in stromal fibronectin and a decrease in epithelial versican in STICs. Our results provide an in-depth picture of the ECM in the benign fallopian tube and identified ECM changes that accompany STIC formation.

PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides

Rush, J. S. — 2021-09-23

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.

A Mechanism for the Rare Fluctuation that Powers Protein Conformational Change

Wu, S. — 2021-09-29

Most functional processes of biomolecules are rare events. Key to a rare event is the rare fluctuation that enables the energy activation process, which powers the system across the activation barrier. But the physical nature of this rare fluctuation and how it enables barrier crossing are unknown. With the help of a novel metric, the reaction capacity pC, that rigorously defines the beginning and parameterizes the progress of energy activation, the rare fluctuation was identified as a special phase-space condition that is necessary and sufficient for initiating systematic energy flow from the non-reaction coordinates into the reaction coordinates. The energy activation of a prototype biomolecular isomerization reaction is dominated by kinetic energy transferring into and accumulating in the reaction coordinates, administered by inertial forces alone. The two major reaction coordinates move in precise synergy, with one acting as a gating mechanism on the other. This mechanism is enabled by the structural features of biomolecules and may the cause of their unique functions that are not possible in small molecules.

Association between Cognitive Function and Large Optic Nerve Cupping, Accounting for Cup-Disc-Ratio Genetic Risk Score

Hallak, J. — 2021-09-30

PurposeTo investigate if accounting for a cup-to-disc ratio (CDR) genetic risk score (GRS) modified the association between large CDR and cognitive function among women. DesignThis was a retrospective study using data from the Womens Health Initiative. MethodsPatients with glaucoma or ocular hypertension were excluded. Large CDR was defined as [≥] 0.6 in either eye. Cognitive function was measured by the Modified Mini-Mental State Examination (3MSE). We used the combined effects from 13 single nucleotide polymorphisms (SNPs) to formulate the GRS for CDR. We used logistic regression to investigate associations between weighted GRS and large CDR, then a linear regression to assess the association between weighted GRS and 3MSE scores, and between weighted GRS, CDR, and 3MSE scores, adjusted for demographic and clinical characteristics. ResultsFinal analyses included 1,196 White women with mean age of 69.60 {+/-} 3.62 years and 7.27% with large CDR. Mean GRS in women with and without large CDR was 1.51 {+/-} 0.31 vs. 1.41 {+/-} 0.36, respectively (p = 0.004). The odds of large CDR for a one unit increase in GRS was 2.30 (95% CI: (1.22, 4.36), p = 0.011). Adding the CDR GRS in the model with CDR and 3MSE, women with large CDR still had statistically significantly lower 3MSE scores than those without large CDR, yielding a predicted mean difference in 3MSE scores of 0.84 (p = 0.007). ConclusionsIndependent of the CDR GRS, women with large CDR had a lower cognitive function.

Directional proton conductance in bacteriorhodopsin is driven by concentration gradient, not affinity gradient

Ren, Z. — 2021-10-04

Many microorganisms can harvest energy from sun light to establish electrochemical potential across cell membrane by pumping protons outward. Light driven proton pumping against a transmembrane gradient entails exquisite electronic and conformational reconfigurations at fs to ms time scales. However, transient molecular events along the photocycle of bacteriorhodopsin are difficult to comprehend from noisy and inconsistent electron density maps obtained from multiple experiments. A major challenge arises from the coexisting intermediate populations as a heterogenous conformational mixture continuously evolves over 13 decades in time. This study reports a meta-analysis of the recent time-resolved datasets collected by several consortia. By resolving structural heterogeneity, this in-depth analysis substantially improves the quality of the electron density maps, and provides a clear visualization of the isolated intermediates from I to M. The earliest photoproducts revealed by the deconvoluted maps suggest that a proton transfer uphill against 15 pH units is accomplished by the same physics governing the tablecloth trick. While the Schiff base is displaced at the beginning of the photoisomerization within ~30 fs, the proton stays due to its inertia. This affinity-independent early deprotonation builds up a steep proton concentration gradient that subsequently drives the directional proton conductance toward the extracellular medium. This mechanism fundamentally deviates from the widely adopted notion on multiple steps of chemical equilibrium driven by light-induced changes of proton affinity. The method of a numerical resolution of concurrent events from mixed observations is also generally applicable. Significance StatementMicroorganisms can exploit solar energy to offset their cellular acidity from the environment by pumping protons outward under light illumination. The ability to transport ions across the cell membrane in response to light makes this family of small transmembrane proteins a highly desirable toolkit in development of new biotechnologies. It is important to understand how these ion pumps operate at the molecular level. This study finds that the outward proton conductance through bacteriorhodopsin, the most studied model system in the class, is driven by a steep concentration gradient of protons established in the light induced process rather than by an affinity gradient previously sought for decades. Summary of RevisionThis is the companion manuscript of another paper already published in PNAS Nexus (Ren, Photoinduced isomerization sampling of retinal in bacteriorhodopsin, PNAS Nexus, 1(3), 2022, 10.1093/pnasnexus/pgac103). The original version of this manuscript was submitted to PNAS Nexus on February 18, 2022. The manuscript was reviewed by three reviewers and the Decision Notification was received on April 5, 2022. I appealed the decision to reject the manuscript on May 28, 2022, and the appeal was accepted. A revised version of the manuscript was submitted on July 25, 2022, with an extensive response to the peer review. The editor sent the revised version and the response to peer review back to the three reviewers. Reviewer 3 declined to review the revised manuscript. The editor extended the invitation to several other scientists to review the revised manuscript. All of them declined to review. The second Decision Notification based on the opinions of Reviewers 1 and 2 was received on September 14, 2022. The revised manuscript, the supplementary materials, and all review documents are listed below in the Table of contents. Second revision is underway.

Pan-cancer organoid validation of tumor outlier chromosomal amplification events

Salahudeen, A. A. — 2021-10-06

Somatic copy number gains are pervasive in many cancer types, yet their roles in oncogenesis are often poorly explored. This lack of understanding is in part due to broad extensions of copy gains across cancer genomes spanning large chromosomal regions, obscuring causal driver loci. Here we employed a multi-tissue pan-organoid modeling approach to validate candidate oncogenic loci identified within pan-cancer TCGA data by the overlap of extreme copy number amplifications with extreme expression dysregulation for each gene. The candidate outlier loci nominated by this integrative computational analysis were functionally validated by infecting cancer type-specific barcoded full length cDNA lentiviral libraries into cognate minimally transformed human and mouse organoids bearing initial oncogenic mutations from esophagus, oral cavity, colon, stomach, pancreas and lung. Presumptive amplification oncogenes were identified by barcode enrichment as a proxy for increased proliferation. Iterative analysis validated DYRK2 at 12q15, encoding a serine-threonine kinase, as an amplified head and neck squamous carcinoma oncogene in p53-/- oral mucosal organoids. Similarly, FGF3, amplified at 11q13 in 41% of esophageal squamous carcinomas, was validated in p53-/- esophageal organoids in vitro and in vivo with pharmacologic inhibition by small molecule and soluble receptor FGFR antagonists. Our studies establish the feasibility of pan-organoid contextual modeling of pan-cancer candidate genomic drivers, enabling oncogene discovery and preclinical therapeutic modeling.

Polyamines mediate enterovirus attachment directly and indirectly through cellular heparan sulfate synthesis.

Hulsebosch, B. M. — 2021-10-31

Productive viral infection begins with attachment to a susceptible cell, and viruses have evolved complex mechanisms to attach to and subsequently enter cells. Prior to engagement with a cellular receptor, viruses frequently interact with nonspecific attachment factors that can facilitate virus-receptor interactions and viral entry. Polyamines, small positively-charged molecules abundant in mammalian cells, mediate viral attachment, though the mechanism was not fully understood. Using the Coxsackievirus B3 (CVB3) enterovirus model system, we show that polyamines mediate viral attachment both directly and indirectly. The polyamine putrescine specifically enhances viral attachment to cells depleted of polyamines. Putrescines positive charge mediates its ability to enhance viral attachment, and polyamine analogs are less efficient at mediating viral attachment. In addition to this direct role of polyamines in attachment, polyamines facilitate the cellular expression of heparan sulfates, negatively-charged molecules found on the cell surface. In polyamine-depleted cells, heparan sulfates are depleted from the surface of cells, resulting in reduced viral attachment. We find that this is due to polyamines role in the process of hypusination of eukaryotic initiation factor 5A, which facilitates cellular translation. These data highlight the important role of polyamines in mediating cellular attachment, as well as their function in facilitating cellular heparan sulfate synthesis.

JAK-STAT Signaling Enables Lineage Plasticity-driven AR Targeted Therapy Resistance

Deng, S. — 2021-11-02

Emerging evidence indicates that various cancers can gain resistance to targeted therapies by acquiring lineage plasticity. Although various genomic and transcriptomic aberrations correlate with lineage plasticity-driven resistance, the molecular mechanisms of acquiring lineage plasticity have not been fully elucidated. Through integrated transcriptomic and single cell RNA-Seq (scRNA-Seq) analysis of more than 80,000 cells, we reveal for the first time that the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling is a crucial executor in promoting lineage plasticity-driven AR targeted therapy resistance in prostate cancer. Ectopic activation of JAK-STAT signaling is specifically required for the AR targeted therapy resistance of subclones expressing multilineage, stem-like and epithelial-to-mesenchymal transition (EMT) lineage transcriptional programs and represents a potential therapeutic target for overcoming AR targeted therapy resistance. One-Sentence SummaryJAK-STAT signaling is a crucial executor in promoting lineage plasticity-driven AR therapy resistance in prostate cancer.

Vitamin D deficiency increases prostatic megalin expression and globulin-bound testosterone import, increasing prostatic androgens in African American men

Garcia, J. — 2021-11-10

Vitamin D deficiency is associated with an increased risk of prostate cancer (PCa) mortality and is hypothesized to contribute to PCa aggressiveness and disparities in African American populations. The prostate epithelium was recently shown to express megalin, an endocytic receptor that internalizes globulin-bound hormones, which suggests regulation of prostate hormone levels, in contrast to the free hormone hypothesis. Here, we demonstrated that megalin imports testosterone bound to sex hormone-binding globulin into prostate cells. Prostatic loss of Lrp2 (megalin) in a mouse model resulted in reduced prostate testosterone and dihydrotestosterone (DHT) levels. Megalin expression was regulated and suppressed by 25-hydroxyvitamin D (25D) in cell lines, patient-derived prostate epithelial cells, and prostate tissue explants, indicating a negative feedback loop. In patent samples, the relationships between hormones support this feedback mechanism, as prostatic DHT levels are higher in African American men and are inversely correlated with serum 25D status. Megalin levels are reduced in localized PCa by the Gleason grade and in patients with future disease recurrence. Our findings suggest that the free hormone hypothesis should be revisited for testosterone and highlight the impact of vitamin D deficiency on prostate androgen levels, which are known drivers of PCa. Thus, we revealed a mechanistic link between vitamin D and PCa disparities observed in African Americans.

The hexokinase ''HKDC1'' interaction with the mitochondria is essential for hepatocellular carcinoma progression

Khan, M. W. — 2021-11-12

Hepatocellular carcinoma (HCC) is a leading cause of death from cancer malignancies. Recently, hexokinase domain containing 1 (HKDC1), was shown to have significant overexpression in HCC compared to healthy tissue. Using in vitro and in vivo tools, we examined the role of HKDC1 in HCC progression. Importantly, HKDC1 ablation stops HCC progression by promoting metabolic reprogramming by shifting glucose flux away from the TCA cycle. Next, HKDC1 ablation leads to mitochondrial dysfunction resulting in less cellular energy which cannot be compensated by enhanced glucose uptake. And finally, we show that the interaction of HKDC1 with the mitochondria is essential for its role in HCC progression, and without this mitochondrial interaction mitochondrial dysfunction occurs. In sum, HKDC1 is highly expressed in HCC cells compared to normal hepatocytes, therefore targeting HKDC1, specifically its interaction with the mitochondria, reveals a highly selective approach to target cancer cells in HCC.

Clonal hematopoiesis in individuals with ANKRD26 or ETV6 germline mutations

Drazer, M. W. — 2021-11-22

Currently, there are at least a dozen recognized hereditary hematopoietic malignancies (HHMs), some of which phenocopy others. Among these, three HHMs driven by germline mutations in ANKRD26, ETV6, or RUNX1 share a phenotype of thrombocytopenia, qualitative platelet defects, and an increased lifetime risk of hematopoietic malignancies (HMs). Prior work has demonstrated that RUNX1 germline mutation carriers experience an elevated lifetime risk (66%) for developing clonal hematopoiesis (CH) prior to age 50. Germline mutations in ANKRD26 or ETV6 phenocopy RUNX1 germline mutations, but no studies have focused on the risk of CH in individuals with germline mutations in ANKRD26 or ETV6. To determine the prevalence of CH in individuals with germline mutations in ANKRD26 or ETV6, we performed next generation sequencing on hematopoietic tissue from twelve individuals with either germline ANKRD26 or germline ETV6 mutations. Each patient had thrombocytopenia but had not developed HMs. Among the seven individuals with germline ANKRD26 mutations, one patient had a CH clone driven by a somatic SF3B1 mutation (p.Lys700Glu). This mutation increased from a variant allele frequency (VAF) of 9.4% at age 56 to 17.4% at age 60. None of the germline ETV6 mutation carriers had evidence of CH at the limits of detection of the NGS assay (5% VAF). Unlike individuals with germline mutations in RUNX1, no individuals under the age of 50 with germline mutations in ANKRD26 or ETV6 had detectable CH. This work demonstrates that ANKRD26 germline mutation carriers, but not ETV6 mutation carriers, experience elevated risk for CH.

A Biophysical Model of Nonquantal Transmission at the Vestibular Hair Cell-Calyx Synapse: KLV currents Modulate Fast Electrical and Slow K+ potentials in the Synaptic Cleft

Govindaraju, A. C. — 2021-11-20

Vestibular hair cells transmit information about head position and motion across synapses to primary afferent neurons. At some of these synapses, the afferent neuron envelopes the hair cell, forming an enlarged synaptic terminal called a calyx. The vestibular hair cell-calyx synapse supports a mysterious form of electrical transmission that does not involve gap junctions termed nonquantal transmission (NQT). The NQT mechanism is thought to involve the flow of ions from the pre-synaptic hair cell to the post-synaptic calyx through low-voltage-activated channels driven by changes in cleft [K+] as K+ exits the hair cell. However, this hypothesis has not been tested with a quantitative model and the possible role of an electrical potential in the cleft has remained speculative. Here we present a computational model that captures salient experimental observations of NQT and identifies overlooked features that corroborate the existence of an electrical potential ({phi}) in the synaptic cleft. We show that changes in cleft{phi} reduce transmission latency and illustrate the relative contributions of both cleft [K+] and{phi} to the gain and phase of NQT. We further demonstrate that the magnitude and speed of NQT depend on calyx morphology and that increasing calyx height reduces action potential latency in the calyx afferent. These predictions are consistent with the idea that the calyx evolved to enhance NQT and speed up vestibular signals that drive neural circuits controlling gaze, balance, and orientation. Significance StatementThe ability of the vestibular system to drive the fastest reflexes in the nervous system depends on rapid transmission of mechanosensory signals at vestibular hair cell synapses. In mammals and other amniotes, afferent neurons form unusually large calyx terminals on certain hair cells, and communication at these synapses includes nonquantal transmission (NQT), which avoids the synaptic delay of quantal transmission. We present a quantitative model that shows how NQT depends on the extent of the calyx covering the hair cell and attributes the short latency of NQT to changes in synaptic cleft electrical potential caused by current flowing through open potassium channels in the hair cell. This previously undescribed mechanism may act at other synapses.

Artificial Intelligence Guided Conformational Mining of Intrinsically Disordered Proteins

Gupta, A. — 2021-11-21

Artificial intelligence recently achieved the breakthrough of predicting the three-dimensional structures of proteins. The next frontier is presented by intrinsically disordered proteins (IDPs), which, representing 30% to 50% of proteomes, readily access vast conformational space. Molecular dynamics (MD) simulations are promising in sampling IDP conformations, but only at extremely high computational cost. Here, we developed generative autoencoders that learn from short MD simulations and generate full conformational ensembles. An encoder represents IDP conformations as vectors in a reduced-dimensional latent space. The mean vector and covariance matrix of the training dataset are calculated to define a multivariate Gaussian distribution, from which vectors are sampled and fed to a decoder to generate new conformations. The ensembles of generated conformations cover those sampled by long MD simulations and are validated by small-angle X-ray scattering profile and NMR chemical shifts. This work illustrates the vast potential of artificial intelligence in conformational mining of IDPs.

OPTN limits herpes stromal keratitis severity and demyelination through negative regulation of IL-17 and hyperinflammatory T-cell response

Ames, J. M. — 2021-11-29

Herpes stromal keratitis (HSK) is a result of the inflammatory sequelae following primary and recurrent Herpes simplex virus type-1 (HSV-1) infections. This pathology is known to be mediated by immunopathogenic T cell responses against viral antigens, however most individuals infected with HSV-1 never exhibit signs of this immunopathology. Recent studies have identified the host restriction factor, optineurin (OPTN), as an inhibitor of viral spread in the central nervous system, protecting hosts from viral encephalopathy. In an HSV-1 corneal infection mouse model on OPTN knockout mice, we assess the contribution of OPTN to ameliorating the clinical manifestations of HSK. We identify that OPTN protects the host from loss of ocular and whisker sensitivity and opacification of the cornea. scRNA-seq of the trigeminal ganglion (TG) reveals that transcription changes to the peripheral neurons and immune cell populations drive the expression of Il-17A in CD4 and CD8 T cells, as well as increased infiltration of T cells into the TG. This leads to demyelination and the observed HSK pathology.

The Atypical Antipsychotic Quetiapine Induces Multiple Antibiotic Resistance in Escherichia coli

Kyono, Y. — 2021-11-30

Atypical antipsychotic (AAP) medication is a critical tool for treating symptoms of psychiatric disorders. While AAPs primarily target dopamine (D2) and serotonin (5HT2A and 5HT1A) receptors, they also exhibit intrinsic antimicrobial activity as an off-target effect. Because AAPs are often prescribed to patients for many years, a potential risk associated with long-term AAP use is the unintended emergence of bacteria with antimicrobial resistance (AMR). Here, we show that exposure to the AAP quetiapine at estimated gut concentrations promotes AMR in Escherichia coli after six weeks. Quetiapine-exposed isolates exhibited an increase in minimal inhibitory concentrations (MICs) for ampicillin, tetracycline, ceftriaxone, and levofloxacin. By whole genome sequencing analysis, we identified mutations in genes that confer AMR, including the repressor for the multiple antibiotic resistance mar operon (marR), and real-time RT-qPCR analysis showed increased levels of marA, acrA, and tolC mRNAs and a reduced level of ompF mRNA in the isolates carrying marR mutations. To determine the contribution of each marR mutation to AMR, we constructed isogenic strains carrying individual mutant marR alleles in the parent background and re-evaluated their resistant phenotypes using MIC and RT-qPCR assays. While marR mutations induced a robust activity of the mar operon, they resulted in only a modest increase in MICs. Interestingly, although these marR mutations did not fully recapitulate the AMR phenotype of the quetiapine-exposed isolates, we show that marR mutations promote growth fitness in the presence of quetiapine. Our findings revealed an important link between the use of AAPs and AMR development in E. coli.

PACAP-PAC1 Receptor Inhibition is Effective in Models of Opioid-Induced Medication Overuse Headache

Bertels, Z. — 2021-12-07

Opioids are regularly prescribed for migraine and can result in medication overuse headache and dependence. We recently showed that pituitary adenylate cyclase activating polypeptide (PACAP) is upregulated following opioid administration or in a model of chronic migraine. The goal of this study was to determine if PACAP was a link between opioid use and headache chronification. We tested the effect of PACAP-PAC1 receptor inhibition in novel models of opioid-exacerbated migraine pain and aura; and examined the co-expression between mu opioid receptor (MOR), PAC1, and PACAP in headache-associated brain and peripheral regions. To model opioid exacerbated migraine pain, mice were injected daily with morphine (10 mg/kg) or vehicle for 11 days. On days 3,5,7,9, and 11 they also received the known human migraine trigger nitroglycerin (0.1 mg/kg) or vehicle. To model opioid exacerbated aura, mice were treated with vehicle or morphine twice daily for 4 days (20 mg/kg on days 1-3, 40 mg/kg on day 4), a well-established paradigm for causing opioid-induced hyperalgesia. On day 5 they underwent cortical spreading depression, a physiological correlate of migraine aura. The effect of the PAC1 inhibitor, M65 (0.1 mg/kg), was tested in these models. Fluorescent in situ hybridization was used to investigate the expression of MOR, PAC1, and PACAP. Only mice treated with combined morphine and nitroglycerin developed chronic cephalic allodynia (n=18/group). M65 reversed this hypersensitivity (n=9/group). Morphine significantly increased the number of CSD events (n=8-9/group); and M65 decreased this exacerbation by morphine (n=8-12/group). PAC1 and/or PACAP were highly co-expressed with MOR, and varied by region (n=6/group). MOR and PACAP were co-expressed in the trigeminal ganglia, while MOR and PAC1 receptor showed near complete overlap in the trigeminal nucleus caudalis and periaqueductal gray. The cortex showed similar cellular co-expression between MOR-PACAP and MOR-PAC1. These results show that opioids facilitate the transition to chronic headache through induction of PACAPergic mechanisms. Antibodies or pharmacological agents targeting PACAP or PAC1 receptor may be particularly beneficial for the treatment of opioid-induced medication overuse headache.

Salmonella enterica serovar Typhimurium chitinases modulate the intestinal glycome and promote small intestinal invasion.

Devlin, J. R. — 2021-12-06

Salmonella enterica serovar Typhimurium (Salmonella) is one of the leading causes of food-borne illnesses worldwide. To colonize the gastrointestinal tract, Salmonella produces multiple virulence factors that facilitate cellular invasion. Chitinases have been recently emerging as virulence factors for various pathogenic bacterial species and the Salmonella genome contains two annotated chitinases: STM0018 (chiA) and STM0233. However, the role of these chitinases during Salmonella pathogenesis is unknown. The putative chitinase STM0233 has not been studied previously and only limited data exists on ChiA. Chitinases typically hydrolyze chitin polymers, which are absent in vertebrates. However, chiA expression was detected in infection models and purified ChiA cleaved carbohydrate subunits present on mammalian surface glycoproteins, indicating a role during pathogenesis. Here, we demonstrate that expression of chiA and STM0233 is upregulated in the mouse gut and that both chitinases facilitate epithelial cell adhesion and invasion. Salmonella lacking both chitinases showed a 70% reduction in invasion of small intestinal epithelial cells in vitro. In a gastroenteritis mouse model, chitinase-deficient Salmonella strains were also significantly attenuated in the invasion of small intestinal tissue. This reduced invasion resulted in significantly delayed Salmonella dissemination to the spleen and the liver, but chitinases were not required for systemic survival. The invasion defect of the chitinase-deficient strain was rescued by the presence of wild-type Salmonella, suggesting that chitinases are secreted. By analyzing N-linked glycans of small intestinal cells, we identified specific N-acetylglucosamine-containing glycans as potential extracellular targets of Salmonella chitinases. This analysis also revealed differential abundance of Lewis X-containing glycans that is likely a result of host cell modulation due to the detection of Salmonella chitinases. Similar glycomic changes elicited by chitinase deficient strains indicate functional redundancy of the chitinases. Overall, our results demonstrate that Salmonella chitinases contribute to intestinal adhesion and invasion through modulation of the host glycome. Author SummarySalmonella Typhimurium infection is one of the leading causes of food-borne illnesses worldwide. In order for Salmonella to effectively cause disease, it has to invade the epithelial cells lining the intestinal tract. This invasion step allows Salmonella to replicate efficiently, causing further tissue damage and inflammation. In susceptible patients, Salmonella can spread past the intestines and infect peripheral organs. It is essential to fully understand the invasion mechanism used by Salmonella to design better treatments for infection. Here, we demonstrate that the two chitinases produced by Salmonella are involved in this invasion process. We show that Salmonella chitinases interact with surface glycans of intestinal epithelial cells and promote adhesion and invasion. Using a mouse infection model, we show that Salmonella chitinases are required for the invasion of the small intestine and enhance the dissemination of Salmonella to other organs. This study reveals an additional mechanism by which Salmonella invades and causes infection.

A multi-layer functional genomic analysis to understand noncoding genetic variation in lipids

Ramdas, S. — 2021-12-08

A major challenge of genome-wide association studies (GWAS) is to translate phenotypic associations into biological insights. Here, we integrate a large GWAS on blood lipids involving 1.6 million individuals from five ancestries with a wide array of functional genomic datasets to discover regulatory mechanisms underlying lipid associations. We first prioritize lipid-associated genes with expression quantitative trait locus (eQTL) colocalizations, and then add chromatin interaction data to narrow the search for functional genes. Polygenic enrichment analysis across 697 annotations from a host of tissues and cell types confirms the central role of the liver in lipid levels, and highlights the selective enrichment of adipose-specific chromatin marks in high-density lipoprotein cholesterol and triglycerides. Overlapping transcription factor (TF) binding sites with lipid-associated loci identifies TFs relevant in lipid biology. In addition, we present an integrative framework to prioritize causal variants at GWAS loci, producing a comprehensive list of candidate causal genes and variants with multiple layers of functional evidence. Two prioritized genes, CREBRF and RRBP1, show convergent evidence across functional datasets supporting their roles in lipid biology.

Molecular mechanism of claudin-15 strand flexibility

Fuladi, S. — 2021-12-09

Claudins are one of the major components of tight junctions that play a key role in formation and maintaining epithelial barrier function. Tight junction strands are dynamic and capable of adapting their structure in response to large-scale tissue rearrangement and cellular movement. Here, we present molecular dynamics simulations of claudin-15 strands of up to 225 nm in length in two parallel lipid membranes and characterize their mechanical properties. The persistence length of claudin-15 strands is comparable with experiments leading to a curvature of 0.12 nm-1 at room temperature. Our results indicate that lateral flexibility of claudin strands is due to an interplay of three sets of interfacial interaction networks between four linear claudin strands in the membranes. In this model, claudins are assembled into interlocking tetrameric ion channels along the strand that slide with respect to each other as the strands curve over sub-micrometer length scales. These results suggest a novel molecular mechanism underlying claudin-15 strand flexibility. It also sheds light on the inter-molecular interactions and their role in maintaining epithelial barrier function.

Structural basis for topological regulation of Tn3 resolvase

Montano, S. P. — 2021-12-08

Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest sitespecific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely-related resolvases from the Tn3 and {gamma}{delta} transposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit protein-DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase.

Physiological activation of the nephron central command drives endogenous kidney tissue regeneration

Gyarmati, G. — 2021-12-09

Tissue regeneration is limited in several organs including the kidney, contributing to the high prevalence of kidney disease globally. However, evolutionary and physiological adaptive responses and the presence of renal progenitor cells suggest existing remodeling capacity. This study uncovered a novel endogenous tissue remodeling mechanism in the kidney that is activated by the loss of body fluid and salt and involves a unique niche of chief cells called macula densa (MD) that control resident progenitor cells via secreted angiogenic, growth and extracellular matrix remodeling factors, cytokines and chemokines. Serial intravital imaging, MD Wnt mouse models and transcriptome analysis provide functional and molecular characterization of this newly identified MD program for kidney regeneration complemented with human and therapeutic translation. The concept that chief cells responding to organ-specific physiological inputs control local progenitors and direct them to remodel or repair tissues may be applicable to other organs and diverse tissue regenerative therapeutic strategies.

Neuroimaging signature associated with symptom exacerbation in early-stage psychosis

Mihaljevic, M. — 2021-12-14

Recent reports have indicated that the occurrence of symptom exacerbation in early-stage psychosis could result in brain changes. Such a symptom exacerbation is frequently called relapse, which underlies a poorer disease outcome. Thus, it is important to identify neuroimaging alterations that are specifically seen in patients who experience relapse in early-stage psychosis. We hypothesized that this specific patient group may be more homogenous in disease-associated signatures likely to be linked to relapse, compared with the overall patient group. Such sub-stratified patient group and neuroimaging signatures would be useful for the biological understanding of relapse. To address this goal, we conducted a cross-sectional study (85 patients with early-stage psychosis and 94 healthy controls) with the use of medical records in a retrospective manner. To define the specific sub-group with the past experience of relapse, we used hospitalization due to psychotic symptom exacerbation, according to many publications that used this factor as a proxy for relapse. By examining resting-state functional connectivity (FC) for the study subjects, we validated our hypothesis and defined 131 FCs possibly associated with relapse. Through these studies, 3 brain regions (the thalamus, precentral gyrus, and dorsal anterior cingulate cortex) were underscored.

Polyclonal lymphoid expansion drives paraneoplastic autoimmunity in neuroblastoma

Rosenberg, M. I. — 2021-12-14

Neuroblastoma is a lethal childhood solid tumor of developing peripheral nerves. Two percent of children with neuroblastoma develop Opsoclonus Myoclonus Ataxia Syndrome (OMAS), a paraneoplastic disease characterized by cerebellar and brainstem-directed autoimmunity, but typically with outstanding cancer-related outcomes. We compared tumor transcriptomes and tumor infiltrating T- and B-cell repertoires from 38 OMAS subjects with neuroblastoma to 26 non- OMAS associated neuroblastomas. We found greater B- and T-cell infiltration in OMAS- associated tumors compared to controls, but unexpectedly showed that both were polyclonal expansions. Tertiary lymphoid structures (TLS) were enriched in OMAS-associated tumors. We identified significant enrichment of the MHC Class II allele HLA-DOB*01:01 in OMAS patients. OMAS severity scores were associated with the expression of several candidate autoimmune genes. We propose a model in which polyclonal autoreactive B lymphocytes act as antigen presenting cells and drive TLS formation, thereby crucially supporting both sustained polyclonal T-cell-mediated anti-tumor immunity and paraneoplastic OMAS neuropathology.

Loss of Depalmitoylation Exaggerates Synaptic Upscaling and Leads to Neuroinflammation in a Lysosomal Storage Disease

Koster, K. P. — 2021-12-17

Palmitoylation and depalmitoylation are the dichotomic processes of lipid modification regulating protein trafficking, recycling, and degradation, thereby controlling proteostasis. Despite our understanding of palmitoylation, depalmitoylation is far less studied. Here, we study a lysosomal depalmitoylating enzyme, palmitoyl-protein thioesterase 1 (PPT1), associated with the devastating neurodegenerative condition CLN1 disease and show that dark-rearing Ppt1-/- mice, which induces synaptic upscaling in vivo, worsen the symptoms. In Ppt1-/- cortical neurons, upscaling induction triggers exaggerated responses of synaptic calcium-permeable AMPA receptors composed of palmitoylated GluA1 subunits. Consequently, Ppt1-/- visual cortex exhibits hypersynchrony in vivo. Remarkably, we also find an overload of palmitoylated A-kinase anchor protein 5 (Akap5) in Ppt1-/- mouse brains, leading to microglial activation through NFAT. These findings indicate Ppt1 acts as a gatekeeper of homeostatic plasticity by regulating the proteostasis of palmitoylated synaptic proteins. Moreover, our results suggest that perturbed depalmitoylation results in neuroinflammation, which is common to neurodegenerative diseases.

Engineered High-Affinity ACE2 Peptide Mitigates ARDS and Death Induced by Multiple SARS-CoV-2 Variants

Zhang, L. — 2021-12-22

Vaccine hesitancy and continuing emergence of SARS-CoV-2 variants of concern that may escape vaccine-induced immune responses highlight the urgent need for effective COVID-19 therapeutics. Monoclonal antibodies used in the clinic have varying efficacies against distinct SARS-CoV-2 variants; thus, there is considerable interest in engineered ACE2 peptides with augmented binding affinities for SARS-CoV-2 Spike protein. These could have therapeutic benefit against multiple viral variants. Using molecular dynamics simulations, we show how three amino acid substitutions in an engineered soluble ACE2 peptide (sACE22.v2.4-IgG1) markedly increase affinity for the SARS-CoV-2 Spike (S) protein. We demonstrate high binding affinity to S protein of the early SARS-CoV-2 WA-1/2020 isolate and also to multiple variants of concern: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) SARS-CoV-2 variants. In humanized K18-hACE2 mice, prophylactic and therapeutic administration of sACE22.v2.4-IgG1 peptide prevented acute lung vascular endothelial injury and lung edema (essential features of ARDS) and significantly improved survival after infection by SARS-CoV-2 WA-1/2020 as well as P.1 variant of concern. These studies demonstrate for the first time broad efficacy in vivo of an ACE2 decoy peptide against multiple SARS-CoV-2 variants and point to its therapeutic potential.

An integrated approach to protein discovery and detection from complex biofluids

Luu, G. T. — 2022-01-04

Ovarian cancer, a leading cause of cancer related deaths among women, has been notoriously difficult to routinely screen for and diagnose early. Researchers and clinicians continue to seek routinely usable, non-invasive, screening methods as early detection significantly improves survival. Biomarker screening is ideal; however, currently available ovarian cancer biomarkers lack desirable sensitivity and specificity. Furthermore, the most fatal forms, high grade serous cancers often originate in the fallopian tube; therefore, sampling from the vaginal environment provides more proximal sources for tumor detection. To address these shortcomings and leverage proximal sampling, we developed an untargeted mass spectrometry microprotein profiling method and identified a signature of cystatin A, validated this protein in an animal model, and sought to overcome the limits of detection inherent to mass spectrometry by demonstrating that cystatin A is present at 100 pM concentrations using a label-free microtoroid resonator. The findings highlight the potential utility for early-stage detection where cystatin A levels would be low. Significance StatementIt is now clear that high-grade serous ovarian cancer can originate in the fallopian tube epithelium. These tumors colonize the ovary and then metastasize throughout the peritoneum. This discovery has raised important, and yet unaddressed, questions how we might be able to detect and screen for this deadly disease for which there is no routine screening. We have leveraged vaginal lavages from a murine model of the disease as a complex biological fluid for untargeted discovery of microproteins using mass. We improved our limits of detection by conjugating a cystatin A antibody to the surface of a microtoroid resonator to allow us to specifically detect cystatin A from vaginal lavages at early time points across biological replicates.

Sequence Coverage Visualizer: A web application for protein sequence coverage 3D visualization

Shao, X. — 2022-01-13

Protein structure is connected with its function and interaction and plays an extremely important role in protein characterization. As one of the most important analytical methods for protein characterization, Proteomics is widely used to determine protein composition, quantitation, interaction, and even structures. However, due to the gap between identified proteins by proteomics and available 3D structures, it was very challenging, if not impossible, to visualize proteomics results in 3D and further explore the structural aspects of proteomics experiments. Recently, two groups of researchers from DeepMind and Baker lab have independently published protein structure prediction tools that can help us obtain predicted protein structures for the whole human proteome. Although there is still debate on the validity of some of the predicted structures, it is no doubt that these represent the most accurate predictions to date. More importantly, this enabled us to visualize the majority of human proteins for the first time. To help other researchers best utilize these protein structure predictions, we present the Sequence Coverage Visualizer (SCV), http://scv.lab.gy, a web application for protein sequence coverage 3D visualization. Here we showed a few possible usages of the SCV, including the labeling of post-translational modifications and isotope labeling experiments. These results highlight the usefulness of such 3D visualization for proteomics experiments and how SCV can turn a regular result list into structural insights. Furthermore, when used together with limited proteolysis, we demonstrated that SCV can help validate and compare different protein structures, including predicted ones and existing PDB entries. By performing limited proteolysis on native proteins at various time points, SCV can visualize the progress of the digestion. This time-series data further allowed us to compare the predicted structure and existing PDB entries. Although not deterministic, these comparisons could be used to refine current predictions further and represent an important step towards a complete and correct protein structure database. Overall, SCV is a convenient and powerful tool for visualizing proteomics results.

Time-lapsed proteomics reveals a role for the novel protein, SNED1, in modulating ECM composition and protein folding

Lee, F. — 2022-01-14

The extracellular matrix (ECM) is a complex and dynamic meshwork of proteins providing structural support to cells. It also provides biochemical signals governing cellular processes including proliferation and migration. Alterations of ECM structure and/or composition has been shown to lead to, or accompany, many pathological processes including cancer and fibrosis. To understand how the ECM contributes to diseases, we first need to obtain a comprehensive characterization of the ECM of tissues and of its changes during disease progression. Over the past decade, mass-spectrometry-based proteomics has become the state-of-the-art method to profile the protein composition of ECMs. However, existing methods do not fully capture the broad dynamic range of protein abundance in the ECM, nor do they permit to achieve the high coverage needed to gain finer biochemical information, including the presence of isoforms or post-translational modifications. In addition, broadly adopted proteomic methods relying on extended trypsin digestion do not provide structural information on ECM proteins, yet, gaining insights into ECM protein structure is critical to better understanding protein functions. Here, we present the optimization of a time-lapsed proteomic method using limited proteolysis of partially denatured samples and the sequential release of peptides to achieve superior sequence coverage as compared to standard ECM proteomic workflow. Exploiting the spatio-temporal resolution of this method, we further demonstrate how 3-dimensional time-lapsed peptide mapping can identify protein regions differentially susceptible to trypsin and can thus identify sites of post-translational modifications, including protein-protein interactions. We further illustrate how this approach can be leveraged to gain insight on the role of the novel ECM protein SNED1 in ECM homeostasis. We found that the expression of SNED1 by mouse embryonic fibroblasts results in the alteration of overall ECM composition and the sequence coverage of certain ECM proteins, raising the possibility that SNED1 could modify accessibility to trypsin by engaging in protein-protein interactions.

Endothelial Nitric Oxide Synthase (eNOS) S1176 phosphorylation status governs atherosclerotic lesion formation

Lee, M. Y. — 2022-01-16

ObjectiveWe have previously demonstrated the in vivo importance of the Akt-eNOS substrate-kinase relationship, as defective postnatal angiogenesis characteristic of global Akt1-null mice is rescued when bred to gain-of-function eNOS S1176D mutant mice. While multiple studies support the vascular protective role of endothelial NO generation, the causal role of Akt1-dependent eNOS S1176 phosphorylation during atherosclerotic plaque formation is not yet clear. Approach & ResultsWe herein bred congenic loss-of-function eNOS S1176A and gain-of-function eNOS S1176D mutant mice to the exacerbated atherogenic Akt1-/-; ApoE-/- double knockout mice to definitively test the importance of Akt-mediated eNOS S1176 phosphorylation during atherogenesis. We find that a single amino acid substitution at the eNOS S1176 phosphorylation site yields divergent effects on atherosclerotic plaque formation, as an eNOS phospho-mimic aspartate (D) substitution at S1176 leads to favorable lipid profiles and decreased indices of atherosclerosis, even when on a proatherogenic Akt1 global deletion background. Conversely, mice harboring an unphosphorylatable mutation to alanine (S1176A) result in increased plasma lipids, increased lesion formation and cellular apoptosis, phenocopying the physiological consequence of eNOS deletion and/or impaired enzyme function. Furthermore, gene expression analyses of whole aortas indicate a combinatorial detriment from NO deficiency and Western Diet challenge, as loss-of-function eNOS SA mice on a Western diet present a unique expression pattern indicative of augmented T-cell activity when compared to eNOS S1176D mice. ConclusionsBy using genetic epistasis approaches, we conclusively demonstrate that Akt-mediated eNOS S1176 phosphorylation and subsequent eNOS activation remains to be the most physiologically relevant method of NO production to promote athero-protective effects.

Syndecan Regulates Cellular Morphogenesis in Cooperation with the Netrin Guidance Pathway and Rho-family GTPases

Dima, R. — 2022-01-14

The establishment of complex cell shapes is essential for specific cellular functions, and thus critical in animal development and physiology. Heparan sulfate proteoglycans (HSPGs) are conserved glycoproteins that regulate interactions between extracellular signals and their receptors, to orchestrate morphogenetic events and elicit cellular responses. Although HSPG-regulated pathways have been implicated in regulating the guidance of neuronal migrations, whether HSPGs regulate earlier aspects of cellular development that dictate cell shape remains unknown. HSPGs consist of a protein core (e.g., Syndecan, Perlecan, Glypican, etc.) with attached heparan sulfate (HS) glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin family. Using mutations in the two C. elegans HS glycosyltransferases genes, rib-1 and rib-2, we reveal that HSPGs control the number of cellular projections in the epithelial excretory canal cell, which can form more than its normal four canals in these mutants. We identify SDN-1/Syndecan as the key HSPG that regulates the number of excretory canal cell projections in a cell-autonomous manner. We also find that Syndecan and guidance receptors for Netrin function in the same pathway to restrict the number of cellular projections. Furthermore, we show that the formation of extra projections in the absence of Syndecan requires the conserved Rho-family GTPases CED-10/Rac and MIG-2/RhoG. Our findings not only contribute to understanding the roles of conserved HSPGs in cellular morphogenetic events, but also reveal the existence of an HSPG-regulated system operating to guarantee that a precise number of cellular projections is established during cell development. Given the evolutionary conservation of developmental mechanisms and the molecules implicated, this work provides information relevant to understanding the cellular and molecular bases of the development of precise cellular morphologies in varied cell types across animals.

Dietary butyrate treatment enhances healthy metabolites by longitudinal untargeted metabolomic analysis in amyotrophic lateral sclerosis mice

Ogbu, D. — 2022-01-18

Microbial metabolites affect the neuron system and muscle cell functions. Amyotrophic Lateral Sclerosis (ALS) is a multifactorial neuromuscular disease. Our previous study has demonstrated elevated intestinal inflammation and dysfunctional microbiome in ALS patients and an ALS mouse model (human-SOD1G93A transgenic mice). However, the metabolites in ALS progression are unknown. Using an unbiased global metabolomic measurement and targeted measurement, we investigated the longitudinal changes of fecal metabolites in the SOD1G93A mice over the course of 13 weeks. We compared the changes of metabolites and inflammatory response in age-matched WT and SOD1G93A mice treated with bacterial product butyrate. We found changes in carbohydrate levels, amino acid metabolism, and formation of gamma-glutamyl amino acids. Shifts in several microbially-contributed catabolites of aromatic amino acids agree with butyrate-induced changes in composition of gut microbiome. Declines in gamma-glutamyl amino acids in feces may stem from differential expression of GGT in response to butyrate administration. Due to signaling nature of amino acid-derived metabolites, these changes indicate changes in inflammation (e.g. histamine) and contribute to differences in systemic levels of neurotransmitters (e.g. GABA, glutamate). Butyrate treatment was able to restore some of the healthy metabolites in ALS mice. Moreover, microglia in the spinal cord were measured by the IBA1 staining. Butyrate treatment significantly suppressed the IBA1 level in the SOD1G93A mice. The serum IL-17 and LPS were significantly reduced in the butyrate treated SOD1G93A mice. We have demonstrated an inter-organ communications link among metabolites, inflammation, and ALS progression, suggesting the potential to use metabolites as ALS hallmarks and for treatment. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/476456v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@16d3e4dorg.highwire.dtl.DTLVardef@1155c05org.highwire.dtl.DTLVardef@14c6155org.highwire.dtl.DTLVardef@18a30d6_HPS_FORMAT_FIGEXP M_FIG We compared the changes of metabolites and inflammatory response in age-matched WT and SOD1G93A mice treated with bacterial product butyrate. Butyrate treatment was able to restore some of the healthy metabolites in ALS mice. Due to signaling nature of amino acid-derived metabolites, these changes indicate changes in inflammation and contribute to differences in systemic levels of neurotransmitters (e.g. GABA, glutamate). Moreover, butyrate treatment significantly suppressed the microglia IBA1 level and aggregated SOD1G93A in the SOD1G93A mice. The inflammatory cytokine, e.g serum IL-17, was significantly reduced in the butyrate treated SOD1G93A mice. We have demonstrated an inter-organ communications link among metabolites, inflammation, and ALS progression, suggesting the potential to use metabolites as ALS hallmarks and for treatment. C_FIG

Knockout of Bbs10 results in lack of cone electrical function and progressive retinal degeneration of rods and cones

Mayer, S. K. — 2022-01-21

Bardet Biedl Syndrome (BBS) is an autosomal recessive disorder caused by mutations in at least 22 different genes. A constant feature is early onset retinal degeneration leading to blindness, with variable central obesity, polydactyly, renal failure, and developmental anomalies. BBS type 10 (BBS10) is a common form caused by mutations in the BBS10 gene encoding a chaperonin-like protein. There are currently no treatments for the progressive vision loss. To aid in treatment development, a BBS10 mouse model was developed by knocking out the Bbs10 gene. Using optical coherence tomography (OCT), electroretinography (ERG), and a visually guided swim assay (VGSA), we demonstrate that Bbs10-/- mice have progressive retinal degeneration. Cone electrical function was absent although cones were anatomically present on histology and retained partial function based on VGSA. The retinal outer nuclear layer (photoreceptor nuclei) progressively thinned as demonstrated on OCT and histology, and rod electrical activity decreased over time on ERG. These phenotypes are more rapidly progressive than retinal degeneration in the Bbs1M390R/M390R knock-in mouse. They are consistent with a cone-rod dystrophy distinct from typical rod-cone degeneration in retinitis pigmentosa and recapitulate aspects of retinal degeneration observed in humans with BBS10. This study has implications for BBS10 gene therapy.

Vitamin D receptor cistrome-transcriptome analyses establishes quantitatively distinct receptor genomic interactions in African American prostate cancer regulated by BAZ1A

Siddappa, M. — 2022-02-01

BackgroundAfrican American (AA) prostate cancer (PCa) appears uniquely sensitive to 1,25(OH)2D3 signaling, compared to European American (EA) PCa, but the extent and impact of vitamin D receptor genomic functions remain poorly defined. ResultsA panel of EA and AA prostate epithelial cells (EA: HPr1-AR, LNCaP, AA: RC43N, RC43T, RC77N, RC77T) were analyzed with RIME to reveal the cell-specific composition of the VDR- complex. 1,25(OH)2D3-dependent ATAC-Seq revealed the greatest impact on nucleosome positioning in RC43N and RC43T, with gain of nucleosome-free at enhancer regions. VDR ChIP-Seq identified stronger and more frequent VDR binding in RC43N and RC43T that was enriched for a larger and distinct motif repertoire, than EA cells. VDR binding significantly overlapped with core circadian rhythm transcription factors in AA cell line models. RNA-Seq also revealed significantly stronger 1,25(OH)2D3 dependent VDR transcriptional responses enriched for circadian rhythm and inflammation networks in AA cells. Whilst RC43N was most responsive, RC43T displayed distorted responses. Significantly reduced BAZ1A/SMARCA5 in AA PCa samples was identified, and restored BAZ1A expression uniquely and significantly increased 1,25(OH)2D3-regulated VDR targets in AA cells. These VDR- dependent cistrome-annotated genes were also uniquely and most significantly identified in three cohorts of AA PCa patients. ConclusionThese data suggest VDR transcriptional control in the prostate is more potent and dynamic in AA men, and primed to govern inflammatory and circadian pathways. Reduced BAZ1A/SMARCA5 expression and/or reduced environmentally-regulated serum vitamin D3 levels suppress these actions. Therefore, the VDR axis lies at the cross-roads of biopsychosocial processes including stress responses, access to quality early detection and treatment, social determinants and that collectively contribute to PCa health disparities.

In vitro effect of a non-immunosuppressive FKBP ligand, FK1706, on SARS-CoV-2 replication in combination with antivirals

Fitzsimmons, W. — 2022-02-04

FKBP, a naturally occurring ubiquitous intracellular protein, has been proposed as a potential target for coronavirus replication. A non-immunosuppressive FKBP ligand, FK1706, was studied in vitro in a Vero cell model to assess potential activity alone and in combination with antivirals against SARS-CoV-2 replication. When combined with remdesivir, synergistic activity was seen (summary synergy score 24.7+9.56). FK1706 warrants in vivo testing as a potential new combination therapeutic for the treatment of COVID-19 infections.

Pancreas resident macrophage-induced fibrosis has divergent roles in pancreas inflammatory injury and PDAC

Baer, J. M. — 2022-02-10

Tissue-resident macrophages (TRMs) are long-lived cells that maintain locally and can be phenotypically distinct from monocyte-derived macrophages (MDMs). However, whether TRMs and MDMs have functional distinction under differing pathologies is not understood. Here, we show a significant portion of macrophages that accumulated during pancreatitis and pancreatic cancer were expanded from TRMs. We further established that pancreas TRMs have a distinct extracellular matrix remodeling phenotype that was critical for maintaining tissue homeostasis during inflammation. Loss of TRMs led to exacerbation of severe pancreatitis and animal death, due to impaired acinar cell survival and recovery. In pancreatitis, TRMs elicited protective effects by triggering the accumulation and activation of fibroblasts, which was necessary for initiating fibrosis as a wound healing response. The same TRM-driven fibrosis, however, drove pancreas cancer pathogenesis and progression. Together, these findings indicate that TRMs play divergent roles in the pathogenesis of pancreatitis and cancer through regulation of stromagenesis.

Dual Inhibition of Cathepsin L and 3CL-Pro by GC-376 Constrains SARS Cov2 Infection Including Omicron Variant

Prabhakar, B. S. — 2022-02-10

Recurrent waves of SARS CoV2 infections remain a major global health concern. Emergence of highly infectious variants with reduced sensitivity to neutralization by vaccines and monoclonal antibodies (mAb) necessitates a deeper understanding of factors involved in SARS CoV2 infections and identification of drug candidates to halt infection. Here, we determined the primacy of endosomal protease cathepsin-L in mediating SARS CoV2 entry and screened a library of well-annotated bioactive compounds for potent cathepsin-L inhibitory activity. Whilst the potent cathepsin-L inhibitors were capable of inhibiting SARS CoV2 entry and cytopathic effect (CPE) in less susceptible cell lines such as human ACE2 expressing 293T cells, these drugs failed to inhibit SARS CoV2 in highly susceptible cell lines such as human TMPRSS2 or human-ACE2-TMPRSS2 overexpressing Vero E6 cells. Only drugs with dual inhibitory effect on both host cathepsin-L and virus 3CL-Protease enzymes such as Z-FA-FMK and GC-376 were capable of inhibiting prototypic (USA-WA1/2020, Lineage A) SARS CoV2 induced CPE in highly susceptible cell lines. Moreover, these drugs inhibited delta (Lineage-B.1.617.2) and omicron (Lineage-B.1.1.529) infection with equal potency showing that the newer mutations harbored in these variants did not affect the mechanism of action of these drugs such as cathepsin-L or 3CL-Pro inhibition. Moreover, our early evidence that 3CL-Pro inhibition can effectively inhibit omicron-induced CPE in highly susceptible cell lines suggests that the recently FDA-approved oral drug, a 3CL-Pro inhibitor which is a combination of nirmatrelvir/ritonavir (Paxlovid) could be effective against omicron variant which shows reduced sensitivity to vaccines and mAb. ImportanceWe report that cathepsin-L and 3CL-Pro as major targets for designing antivirals against SARS CoV2. Dual inhibition of cathepsin-L and 3CL-Pro by GC-376 renders it effective in inhibiting SARS CoV2-induced cytopathic effect in highly susceptible cell lines. Moreover, this candidate drug is equally effective against prototypic SARS CoV2 lineage A and emerging variants such as delta and omicron which show reduced sensitivity to vaccines and monoclonal antibodies. Given the recent wave of SARS CoV2 omicron variant infection around the world, and 3CL-Pro inhibitor nirmatrelvir is one of the components of the FDA-approved Paxlovid, our findings are timely, important and should be of broad interest.

Sequence-Dependent Backbone Dynamics of Intrinsically Disordered Proteins

Dey, S. — 2022-02-12

For intrinsically disordered proteins (IDPs), a pressing question is how sequence codes for function. Dynamics serves as a crucial link, reminiscent of the role of structure in sequence-function relations of structured proteins. To define general rules governing sequence-dependent backbone dynamics, we carried out long molecular dynamics simulations of eight IDPs. Blocks of residues exhibiting large amplitudes in slow dynamics are rigidified by local inter-residue interactions or secondary structures. A long region or an entire IDP can be slowed down by long-range contacts or secondary- structure packing. On the other hand, glycines promote fast dynamics and either demarcate rigid blocks or facilitate multiple modes of local and long-range inter-residue interactions. The sequence-dependent backbone dynamics endows IDPs with versatile response to binding partners, with some blocks recalcitrant while others readily adapting to intermolecular interactions.

Constitutively active STAT5b feminizes mouse liver gene expression

Lau-Corona, D. — 2022-02-14

STAT5 is an essential transcriptional regulator of the sex-biased actions of growth hormone (GH) in the liver. Delivery of constitutively active STAT5 (STAT5CA) to male mouse liver using an engineered adeno-associated virus with high tropism for the liver is shown to induce widespread feminization of the liver, with extensive induction of female-biased genes and repression of male-biased genes, largely mimicking results obtained when male mice are given GH as a continuous infusion. Many of the gene expression changes observed were associated with STAT5 binding to liver chromatin, supporting the proposed direct role of persistently active STAT5 in continuous GH-induced liver feminization. The feminizing effects of STAT5CA were dose-dependent; moreover, at higher levels, overexpression of STAT5CA resulted in some histopathology not seen in continuous GH-infused male liver, including hepatocyte hyperplasia and distorted liver architecture. These findings establish that the persistent activation of STAT5 by GH that characterizes female liver is by itself sufficient to account for the female-biased expression of a majority of female-biased genes. Moreover, histological changes seen when STAT5CA is overexpressed highlight the importance of carefully evaluating such effects before considering such STAT5 derivatives for therapeutic use in treating liver disease.

scDVF: Data-driven Single-cell Transcriptomic Deep Velocity Field Learning with Neural Ordinary Differential Equations

Chen, Z. — 2022-02-19

Recent advances in single-cell RNA sequencing technology have provided unprecedented opportunities to simultaneously measure the gene expression profile and transcriptional velocity of individual cells, enabling us to sample gene regulatory network dynamics along developmental trajectories. However, traditional methods have faced challenges in modeling gene expression dynamics within individual cells due to sparse, non-linear (e.g., obligate heterodimer transcription factors), and high-dimensional measurements. Here, we present DeepVelo, a neural-network-based ordinary differential equation model that can learn non-linear, high-dimensional single-cell transcriptome dynamics and describe continuous gene expression changes within individual cells across time. We applied DeepVelo to multiple published datasets from different technical platforms and demonstrated its utility to 1) formulate transcriptome dynamics on different timescales, 2) measure the instability of cell states, and 3) identify developmental driver genes upstream of a signaling cascade. Benchmarking against state-of-the-art methods shows that DeepVelo can improve velocity field representation accuracy by at least 50% in out-of-sample cells. Further, perturbation studies revealed that single-cell dynamical systems may exhibit properties similar to those of chaotic systems. In summary, DeepVelo allows for the data-driven discovery of differential equations that delineate single-cell transcriptome dynamics. TeaserEmbedding neural networks into ordinary differential equations to model gene expression changes within single cells across time.

Insights into the ribosome function from the structures of non-arrested ribosome nascent chain complexes.

Syroegin, E. A. — 2022-02-21

During protein synthesis, the growing polypeptide chain threads through the nascent peptide exit tunnel that spans the body of the large ribosomal subunit while simultaneously acting as a modulator of ribosomal activity by itself or by sensing various small molecules, such as metabolites or antibiotics appearing in the tunnel. While arrested ribosome nascent chain complexes (RNCCs) have been extensively studied structurally, little attention has been given to the RNCCs that represent the functionally active state of the ribosome. This is in part due to the lack of a simple and reliable procedure for the large-scale preparation of peptidyl-tRNAs. Here we report a new chemoenzymatic approach based on native chemical ligation reaction for the facile synthesis of stably linked peptidyl-tRNAs that were used to determine several structures of RNCCs in the functional pre-attack state of the peptidyl transferase center (PTC) at the highest resolution available to date. These structures reveal a previously unknown role of the ribosome in stabilization of the growing polypeptide within the PTC and suggest an extended entropic trap model that mechanistically rationalizes how ribosome acts with comparable efficiencies upon a multitude of possible growing peptides having various sequences. Our structures also provide new insights into the mechanism of PTC functioning and explain what makes ribosome a versatile catalyst.

Shifting a Cellular Metabolic Landscape Identifies a Refractory Environment for Flavivirus Replication

Gullberg, R. C. — 2022-02-21

Host-targeted therapeutics to control viral infection are gaining prominence given the vulnerability of viral replication at select host-interaction points and the limited possibility of developing drug resistant mutants. Nevertheless, the chemical and biological impact of many host-targeted therapeutics on both the cell and virus has not been elucidated and remains a key complication. Previously, it has been demonstrated that inhibition of fatty acid metabolism has significant antiviral potential. Here, we use a multidisciplinary approach to demonstrate how inhibition of fatty acid biosynthesis creates a metabolically refractory environment that drives viral dependence on alternate metabolic pathways for survival. By profiling the global metabolic landscape following inhibition of fatty acid biosynthesis, we identified additional biochemical pathways that, when inhibited in combination with fatty acid biosynthesis, displayed increased antiviral potential. Our studies also demonstrated that there was a direct link between changes in cellular chemical composition and the ultrastructural membrane architecture induced by viral gene products. Utilizing inhibitors to change these metabolic environments significantly impacted early viral replication and disrupted the membrane architecture critical for the viral life cycle. Here, we have defined at a molecular level how shifting metabolic landscapes can be exploited to identify combinations of therapeutics that have a greater antiviral effect. Author SummaryDengue viruses are transmitted by Aedes aegypti mosquitoes which are prevalent in the tropical and subtropical regions of the world. These viruses cause over 350 million infections annually. There are no antivirals to combat infection and the only vaccine available is suboptimal. Since these viruses are obligate pathogens, they hijack lipid metabolic pathways in host cells to drive new lipid synthesis critically required for their replication. Mechanisms of how lipid synthesis impacts viral replication is unknown. These viruses also rearrange cellular membranes to form platforms for assembly of viral replication complexes. Here, for the first time, we show that virus-hijacking of de novo fatty acid biosynthesis pathways is required for the formation of membranous replication platforms and if inhibited disrupted synthesis of replicative form viral RNA. Importantly, these inhibitors drastically rearranged the metabolic landscape of the cell resulting in an activation of compensatory nucleotide synthesis pathways that allowed the virus to survive at a low level through the inhibition. However, if both pathways were inhibited in combination, infectious virus release was reduced to below detection limits. The study demonstrates how understanding the metabolic landscape altered by specific inhibitors can lead to the discovery of compensatory metabolic pathways and targets that in combination can enhance intervention efficacy.

Tango for Two: Modeling Synchronization and Social Interaction in Turn-Taking

Begel, V. — 2022-02-27

Important individual differences are observed in peoples abilities to synchronize their body movements with regular auditory rhythms. We investigate whether synchronizing with a regular auditory cue is affected by each persons spontaneous production rate (SPR) and by hearing a partners synchronization in a social context. Musically trained and untrained participants synchronized their tapping with an auditory cue presented at different rates (their own SPR or their partners SPR) and in a Solo or Joint (turn-taking) condition. Linear and nonlinear oscillator models were fit to participants mean asynchronies (signed timing differences between the cued onsets and taps). In Joint turn-taking, participants synchrony was increased when the auditory signal was cued at the participants own SPR, compared with their partners SPR; in contrast, synchronization did not differ across rates in the Solo condition. Asynchronies in the Joint task became larger as the difference between partners spontaneous rates increased; the increased asynchronies were driven by the faster partner who did not slow down to match the rate of their slower partner. Nonlinear delay-coupled models (with time delay, coupling strength, and intrinsic frequency) outperformed linear models (intrinsic frequency only) in accounting for tappers synchronization adjustments. The nonlinear models coupling value increased for musically trained participants, relative to untrained participants. Overall, these findings suggest that both intrinsic differences in partners spontaneous rates and the social turn-taking context contribute to the range of synchrony in the general population. Delay-coupled models are capable of capturing the wide range of individual differences in auditory-motor synchronization.

4D Cell-Condensate Bioprinting

Ding, A. — 2022-03-02

4D bioprinting techniques that facilitate formation of shape-changing scaffold-free cell condensates with prescribed geometries have yet been demonstrated. Here, a simple yet novel 4D bioprinting approach is presented that enables formation of a shape-morphing cell condensate-laden bilayer system comprised of an actuation layer and a cell condensate-supporting microgel (MG) layer. The strategy produces scaffold-free cell condensates which morph over time into predefined complex shapes. With a sequential printing (i.e., MG printing first onto the preformed actuation hydrogel layer and cell-only printing inside the pre-printed MG construct second), cell condensate-laden bilayers with specific geometries are readily fabricated and can be further UV-crosslinked to form strong interlayer adhesion. Since the bilayers have tunable deformability and MG degradation can be tailored, this enables controllable morphological transformations and on-demand liberation of cell condensates. With this system, large cell condensate-laden constructs with various complex shapes were obtained through predefined conformational conversions. As a proof-of-concept study, the formation of the letter "C" and helix-shaped robust cartilage-like tissues differentiated from human mesenchymal stem cells (hMSCs) was demonstrated. This new system brings about a new versatile 4D bioprinting platform idea that is anticipated to broaden and facilitate the applications of cell condensation-based 4D bioprinting.

Nucleocapsid-specific humoral responses improve the control of SARS-CoV-2

Dangi, T. — 2022-03-09

The spike protein of SARS-CoV-2 is a critical antigen present in all approved SARS-CoV-2 vaccines. This surface viral protein is also the target for all monoclonal antibody therapies, but it is unclear whether antibodies targeting other viral proteins can also improve protection against COVID-19. Here, we interrogate whether nucleocapsid-specific antibodies can improve protection against SARS-CoV-2. We first immunized mice with a nucleocapsid-based vaccine, and then transferred sera from these mice into naive mice. On the next day, the recipient mice were challenged intranasally with SARS-CoV-2 to evaluate whether nucleocapsid-specific humoral responses affect viral control. Interestingly, mice that received nucleocapsid-specific sera exhibited enhanced control of a SARS-CoV-2 infection. These findings provide the first demonstration that humoral responses specific to an internal coronavirus protein can help clear infection, warranting the inclusion of other viral antigens in next-generation SARS-CoV-2 vaccines and providing a rationale for the clinical evaluation of nucleocapsid-specific monoclonals to treat COVID-19. HighlightsA SARS-CoV-2 nucleocapsid vaccine elicits robust nucleocapsid-specific antibody responses. This nucleocapsid vaccine generates memory B cells (MBC). Nucleocapsid-specific humoral responses do not prevent SARS-CoV-2 infection. Nucleocapsid-specific humoral responses help control a SARS-CoV-2 infection.

Intrathecal tracer dispersion is accelerated by high volume injection and natural micromixing

Ayansiji, A. O. — 2022-03-25

BackgroundTraditionally, there is a widely held belief that drug dispersion after intrathecal (IT) delivery is confined locally near the injection site. We posit that high volume infusions can overcome this perceived limitation of IT administration. MethodsTo test our hypothesis, subject-specific deformable phantom models of the human central nervous system were manufactured so that tracer infusion could be realistically replicated in vitro over the entire physiological range of pulsating cerebrospinal fluid (CSF) amplitudes and frequencies. Dispersion of IT injected tracers was studied systematically with high-speed optical methods to determine the relative impact of injection parameters including infusion volume, flow rate, catheter configurations and natural CSF oscillations. ResultsOptical imaging analysis of high-volume infusion experiments showed that tracer spreads quickly throughout the spinal subarachnoid space (SAS), reaching the cervical region in less than ten minutes. The experimentally observed biodispersion is much faster than suggested by prior theories (Taylor-Aris-Watson TAW dispersion). Our experiments indicate that micro-mixing patterns induced by oscillatory CSF flow around microanatomical features such as nerve roots significantly accelerate solute transport. Strong micro mixing effects due to anatomical features in the spinal subarachnoid space were found to be active in intrathecal drug administration but were not considered in prior dispersion theories. Their omission explains why prior models developed in the engineering community are poor predictors for IT delivery. ConclusionOur experiments support the feasibility of targeting large sections of the neuroaxis or brain utilizing high-volume IT injection protocols. The experimental tracer dispersion profiles acquired with an in vitro human CNS analog informed a new predictive model of tracer dispersion as a function of physiological CSF pulsations and adjustable infusion parameters. The ability to predict spatiotemporal dispersion patterns is an essential prerequisite for exploring new indications of IT drug delivery that targets specific regions in the central nervous system (CNS) or the brain.

Intestinal epithelial Axin1 deficiency protects against colitis via altered gut microbiota

Garrett, S. — 2022-03-26

Background and AimsIntestinal homeostasis is maintained by specialized host cells and the gut microbiota. Wnt/{beta}-catenin signaling is essential for gastrointestinal development and homeostasis, and its dysregulation has been implicated in inflammation and colorectal cancer. Axin1 negatively regulates activated Wnt/{beta}-catenin signaling, but little is known regarding its role in regulating host-microbial interactions in health and disease. Here, we aim to demonstrate that intestinal Axin1 determines gut homeostasis and host response to inflammation. MethodsThe expression of Axin1 was analyzed in human IBD datasets. To explore the effects and mechanism of intestinal Axin1 in regulating intestinal homeostasis and colitis, we generated mouse models with Axin1 conditional knockout in intestinal epithelial (Axin1{Delta}IEC) and Paneth cells (Axin1{Delta}PC) to compare with control (Axin1LoxP) mice. ResultsWe found increased Axin1 expression in the colonic epithelium of human IBD. Axin1{Delta}IEC mice exhibited altered goblet cell spatial distribution, Paneth cell morphology, reduced lysozyme expression, and enriched Akkermansia muciniphila. Absence of intestinal epithelial and Paneth cell Axin1 decreased susceptibility to DSS-induced colitis in vivo. Axin1{Delta}IEC and Axin1{Delta}PC became more susceptible to DSS-colitis after cohousing with control mice, suggesting the non-colitogenic effect is driven by the gut microbiota. ConclusionWe found loss of intestinal Axin1 protects against colitis, which is likely driven through Paneth cell Axin1 and the microbiota. Our study demonstrates a novel role of Axin1 in mediating intestinal homeostasis and the microbiota. Further mechanistic studies using specific Axin1 mutations elucidating how Axin1 modulates microbiome and host inflammatory response, will provide new therapeutic strategies for human IBD. What you Need to Know1. Background and ContextWnt/beta-catenin is a fundamental molecular pathway that affects intestinal proliferation and differentiation. Axin1 negatively regulates activated Wnt/{beta}-catenin signaling, but little is known regarding its role in the microbiome. Dysfunction of Wnt/beta-catenin was reported in human inflammatory bowel disease (IBD) and Axin1 serum level was elevated in patients with UC. 2. New FindingsWe found increased Axin1 expression at both the mRNA and protein level in human IBD. Specifically, we identified increased Axin1 expression positive correlated with pro-inflammatory cytokines IL-6 and TNF- in CD. Our study, for the first time, identifies links between the gut microbiota and intestinal Axin1 in intestinal inflammation through utilization of innovative deletion mouse models in intestinal epithelium and Paneth cells. Loss of intestinal Axin1 plays a novel role in intestinal inflammation by altering the Paneth cells and microbiome (e.g., enriched Akkermansia mucinlphila). Our study has provided insights into the molecular mechanism that might contribute to IBD, especially the novel role of Paneth cell Axin1 in colitis. 3. LimitationsThere are no human or mice studies assessing the role of intestinal epithelial and Paneth cell Axin1 in inflammation and the microbiome. 4. ImpactFurther explorations of the gut microbiota and Axin1 interaction as we report will provide novel mechanistic strategies for therapeutic approaches for human IBD by targeting intestinal Axin1 and Axin1-associated microbiome.

An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2

Zhang, L. — 2022-03-28

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) glycoprotein neutralize infection and are efficacious for the treatment of mild-to-moderate COVID-19. However, SARS-CoV-2 variants have emerged that partially or fully escape monoclonal antibodies in clinical use. Notably, the BA.2 sublineage of B.1.1.529/omicron escapes nearly all monoclonal antibodies currently authorized for therapeutic treatment of COVID-19. Decoy receptors, which are based on soluble forms of the host entry receptor ACE2, are an alternative strategy that broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE22.v2.4-IgG1 was previously shown to be effective in vivo against SARS-CoV-2 variants when administered intravenously. Here, the inhalation of sACE22.v2.4-IgG1 is found to increase survival and ameliorate lung injury in K18-hACE2 transgenic mice inoculated with a lethal dose of the virulent P.1/gamma virus. Loss of catalytic activity reduced the decoys therapeutic efficacy supporting dual mechanisms of action: direct blocking of viral S and turnover of ACE2 substrates associated with lung injury and inflammation. Binding of sACE22.v2.4-IgG1 remained tight to S of BA.1 omicron, despite BA.1 omicron having extensive mutations, and binding exceeded that of four monoclonal antibodies approved for clinical use. BA.1 pseudovirus and authentic virus were neutralized at picomolar concentrations. Finally, tight binding was maintained against S from the BA.2 omicron sublineage, which differs from S of BA.1 by 26 mutations. Overall, the therapeutic potential of sACE22.v2.4-IgG1 is further confirmed by inhalation route and broad neutralization potency persists against increasingly divergent SARS-CoV-2 variants.

Super-Resolution NMR Spectroscopy using the Intersection of Non-Redundant Information on Resonance Groups

Lorieau, J. L. — 2022-04-12

The resolution of spectra is a major limitation in the application of Nuclear Magnetic Resonance (NMR) spectroscopy to large and complex molecular systems. In this report, we introduce a technique to enhance the resolution of NMR spectra beyond the intrinsic limitations of a spectrometer for a single spectrum by using the Intersection of Non-Redundant Information on Resonance Groups (INIR). With INIR, we reconstruct 900-MHz (21.1T) spectra from a 500-MHz (11.7T) NMR spectrometer, which compare favorably to experimental 900-MHz spectra. INIR holds promise in significantly enhancing the resolution of NMR spectra and in extending the size and complexity of molecules studied by NMR.

The yeast claudin Dcv1 is important for demarcating the front domain of pheromone-responding yeast cells

Sukumar, M. — 2022-04-19

Cell polarization in response to chemical gradients is important in development and homeostasis across eukaryota. Chemosensing cells orient toward or away from gradient sources by polarizing along a front-rear axis. Using the chemotropic mating response of the budding yeast S. cerevisiae as a model of environmentally-induced cell polarization, we found that Dcv1, a claudin homolog, is a determinant of front-rear polarity. Although Dcv1 localized uniformly on the plasma membrane (PM){dagger} of vegetative cells, it was confined to the rear of cells responding to pheromone, away from the pheromone receptor and mating projection. Deletion of DCV1 conferred mislocalization of sensory, polarity, and trafficking proteins, as well as the PM lipids ergosterol, phosphatidylinositol-4,5-bisphosphate (PIP2), and phosphatidylserine (PS). These phenotypes correlated with defects in pheromone-gradient tracking and cell fusion. We propose that the novel claudin-like and rear-domain protein, Dcv1, demarcates the mating-specific front domain primarily by restricting PM lipid distribution. Consistent with this hypothesis, a mutation that blocks ergosterol biosynthesis partially phenocopies dcv1{Delta}. Summary statementThe yeast claudin Dcv1 facilitates the proper localization of plasma membrane lipids and proteins that are required for front-rear polarity, efficient chemotropism, and cell fusion.

Simple within-stride changes in treadmill speed can drive selective changes in human gait symmetry

Browne, M. G. — 2022-04-22

BackgroundMillions of people worldwide are affected by clinical conditions that result in gait asymmetry (e.g., stroke, cerebral palsy, lower limb amputation). There is a need for customizable rehabilitation approaches that can flexibly target different aspects of gait asymmetry with minimal need for specialized equipment. Here, we studied how simple within-stride changes in treadmill speed could drive selective, predictable changes in human gait symmetry. MethodsIn Experiment 1, ten healthy young adults walked on an instrumented treadmill with and without a closed-loop controller engaged. The controller changed the treadmill speed to 1.50 m/s or 0.75 m/s depending on whether the right or left leg generated propulsive (i.e., forward-directed) ground reaction forces, respectively. In Experiment 2, a separate group of ten healthy young adults walked on the treadmill with and without an open-loop controller engaged. This controller changed the treadmill speed to 1.50 m/s or 0.75 at a prescribed time interval. We used a metronome to guide the participants to step at a series of different time points relative to the controller-driven speed change. We collected kinematic and kinetic data in Experiments 1 and 2. ResultsIn Experiment 1, participants walked with asymmetric kinematics and ground reaction forces when the closed-loop controller was engaged. The leg that accelerated during propulsion (right leg) showed a smaller leading limb angle and a larger trailing limb angle than the leg that decelerated during propulsion (left leg). The right leg also generated smaller propulsive forces than the left leg. In Experiment 2, the patterns of asymmetry in spatiotemporal gait parameters, kinematics, and ground reaction forces depended on the timing of the speed change within the gait cycle. Step times, leading limb angles, and peak propulsion became asymmetric when the treadmill speed changed early in stance. When the treadmill speed changed later in stance, step lengths, step times, and propulsion impulses became asymmetric. ConclusionsSimple manipulations of treadmill speed can drive predictable, selective changes in human gait symmetry. Future work will explore this customizable technique as a potential approach for restoring gait symmetry in clinical populations.

Optimizing Oscillators for Specific Tasks Predicts Preferred Biochemical Implementations

Agrahar, C. — 2022-04-26

Oscillatory processes are used throughout cell biology to control time-varying physiology including the cell cycle, circadian rhythms, and developmental patterning. It has long been understood that free-running oscillations require feedback loops where the activity of one component depends on the concentration of another. Oscillator motifs have been classified by the positive or negative net logic of these loops. However, each feedback loop can be implemented by regulation of either the production step or the removal step. These possibilities are not equivalent because of the underlying structure of biochemical kinetics. By computationally searching over these possibilities, we find that certain molecular implementations are much more likely to produce stable oscillations. These preferred molecular implementations are found in many natural systems, but not typically in artificial oscillators, suggesting a design principle for future synthetic biology. Finally, we develop an approach to oscillator function across different reaction networks by evaluating the biosynthetic cost needed to achieve a given phase coherence. This analysis predicts that phase drift is most efficiently suppressed by delayed negative feedback loop architectures that operate without positive feedback. PACS numbers47.15.-x

Gestational day 12 moderate prenatal alcohol exposure produces sex-specific social impairments and attenuates prelimbic excitability and amygdala-cortex modulation of adult social behavior.

Przybysz, K. R. — 2022-05-04

Lifelong social impairments are common in individuals with prenatal alcohol exposure (PAE), and preclinical studies have identified gestational day (G)12 as a vulnerable timepoint for producing social deficits following binge-level PAE. While moderate (m)PAE also produces social impairments, the long-term neuroadaptations underlying them are poorly understood. Activity of the projection from the basolateral amygdala to the prelimbic cortex (BLA[->]PL) leads to social avoidance, and the PL alone is implicated in negative social behaviors, making each of these potential candidates for the neuroadaptations underlying mPAE-induced social impairments. To examine this, we first established that G12 mPAE produced sex-specific social impairments lasting into adulthood. We then chemogenetically inhibited the BLA[->]PL using Clozapine N-Oxide (CNO) during adult social testing. This revealed that CNO reduced social investigation in control males, but had no effect on mPAE males or females of either exposure, indicating that mPAE attenuated the role of this projection in regulating male social behavior and highlighting one potential mechanism by which mPAE affects male social behavior more severely. Using whole-cell electrophysiology, we also examined mPAE-induced changes to PL pyramidal cell physiology and determined that mPAE reduced the excitability of these cells, likely due to increased suppression by inhibitory interneurons. Overall, this work identified two mPAE-induced neuroadaptations that last into adulthood and which may underlie the sexspecific vulnerability to mPAE-induced social impairments. Future research is necessary to expand upon how these circuits modulate both normal and pathological social behavior, and to identify sex-specific mechanisms leading to differential vulnerability in males and females.

Human papilloma virus E6 regulates therapy responses in oropharyngeal cancer by repressing the PGC-1α/ERRα axis

Sannigrahi, M. K. — 2022-05-05

Therapy with radiation plus cisplatin kills human papilloma virus-related (HPV+) oropharyngeal squamous cell carcinomas (OPSCCs) by increasing reactive oxygen species beyond cellular antioxidant capacity. To explore why some patients fail these standard treatments, we evaluated whether the variation in HPV oncoprotein levels among HPV+ OPSCCs impacts mitochondrial metabolism, a source of antioxidant capacity. In cell line and patient-derived xenograft models, levels of HPV full-length E6 (fl-E6) inversely correlated with oxidative phosphorylation, antioxidant capacity, and therapy resistance, and fl-E6 was the only HPV oncoprotein to display such correlation. Ectopically expressing fl-E6 in models with low levels reduced mitochondrial mass, depleted antioxidant capacity, and sensitized to therapy. In this setting, fl-E6 repressed the PGC-1/ERR pathway for mitochondrial biogenesis by reducing p53-dependent PGC-1 transcription. Concordant observations were made in three clinical cohorts, where expression of mitochondrial components was higher in tumors of patients with reduced survival. These tumors contained the lowest fl-E6 levels, highest p53 target gene expression, and an activated PGC-1/ERR pathway. Our findings demonstrate that E6 can potentiate treatment responses by depleting mitochondrial antioxidant capacity and provide evidence for low E6 negatively impacting patient survival. E6s interaction with the PGC-1/ERR axis has implications for predicting and targeting treatment resistance in OPSCC.

Inhibition of Aromatase by Hops, Licorice Species, and their bioactive compounds in Postmenopausal Breast Tissue

Hajirahimkhan, A. — 2022-05-08

Breast cancer risk continues to rise post menopause. Endocrine therapies are employed to prevent postmenopausal breast cancer in high-risk women. However, their adverse effects have reduced acceptability and overall success in cancer preventionatural products such as hops (Humulus lupulus) and pharmacopoeial licorice (Glycyrrhiza) species, which are used for managing menopausal symptoms, have demonstrated estrogenic and chemopreventive properties. Their beneficial effects on aromatase activity and expression as important factors in postmenopausal breast carcinogenesis are understudied. The presented data show that Gycyrrhiza inflata (GI) has the highest aromatase inhibition potency among these plants. Moreover, phytoestrogens such as 8-prenylnaringenin from hops as well as liquiritigenin and 8-prenylapigenin from licorice are shown to be potent bioactives, in line with computational docking studies. 8-Prenylnaringenin, GI extract, liquiritigenin, and licochalcone A all suppress aromatase expression in postmenopausal womens breast tissue. Collectively, these data suggest that these natural products may have breast cancer prevention potential for high-risk postmenopausal women.

The proteomic and transcriptomic landscapes altered by Rgg2/3 activity in Streptococcus pyogenes

Rued, B. E. — 2022-05-07

Streptococcus pyogenes, otherwise known as Group A Streptococcus (GAS), is an important and highly adaptable human pathogen with the ability to cause both superficial and severe diseases. Understanding how S. pyogenes senses and responds to its environment will likely aid in determining how it causes a breadth of diseases. One regulatory network involved in GASs ability to sense and respond to the changing environment is the Rgg2/3 quorum sensing (QS) system, which responds to metal and carbohydrate availability and regulates changes to the bacterial surface. To better understand the impact of Rgg2/3 QS on S. pyogenes physiology, we performed RNA-seq and TMT-LC-MS/MS analysis on cells in which this system was induced or disrupted. Primary findings confirmed that pheromone stimulation in wildtype cultures is limited to the induction of operons whose promoters contain previously determined Rgg2/3 binding sequences. However, supplementing exogenous pheromone to a deletion mutant of rgg3, a strain that endogenously produces elevated amounts of pheromone, led to extended alterations of the transcriptome and proteome, ostensibly by stress-induced pathways. Under such exaggerated pheromone conditions ({Delta}rgg3+SHP), a connection was identified between Rgg2/3 and the stringent response. Mutation of relA, the bifunctional guanosine tetra- and penta-phosphate nucleoside synthetase/hydrolase, and alarmone synthase genes sasA and sasB, impacted culture doubling times and disabled induction of Rgg2/3 in response to mannose, while manipulation of Rgg2/3 signaling modestly altered nucleotide levels. Our findings indicate that excessive pheromone production or exposure places stress on GAS resulting in an indirect altered proteome and transcriptome beyond primary pheromone signaling. IMPORTANCEStreptococcus pyogenes causes several important human diseases. This study evaluates how the induction or disruption of a cell-cell communication system alters S. pyogeness gene expression and, in extreme conditions, its physiology. Using transcriptomic and proteomic approaches, the results define the pheromone-dependent regulon of the Rgg2/3 quorum sensing system. In addition, we find that excessive pheromone stimulation, generated by genetic disruption of the system, leads to stress responses that are associated with the stringent response. Disruption of this stress response affects the ability of the cell-cell communication system to respond under certain conditions. These findings assist in the determination of how S. pyogenes is impacted by and responds to non-traditional sources of stress.

Quorum sensing regulation of a major facilitator superfamily transporter affects multiple streptococcal virulence factors

Chang, J. C. — 2022-05-07

Cell-cell signaling mediated by Rgg-family transcription factors and their cognate pheromones is conserved in Firmicutes, including all streptococci. In Streptococcus pyogenes, or Group A strep, one of these systems, the Rgg2/3 quorum sensing (QS) system, has been shown to regulate phenotypes including cellular aggregation and biofilm formation, lysozyme resistance, and macrophage immunosuppression. Here, we show that the abundance of several secreted virulence factors (streptolysin O, SpyCEP, and M protein) decreases upon induction of QS. The main mechanism underlying the changes in protein levels appears to be transcriptional, occurs downstream of the QS circuit and is dysregulated by the deletion of an Rgg2/3 QS-regulated major facilitator superfamily (MFS) transporter. Additionally, we identify this MFS transporter as the factor responsible for a previously observed increase in aminoglycoside sensitivity in QS-induced cells. ImportanceThe production of virulence factors is a tightly regulated process in bacterial pathogens. Efforts to elucidate the mechanisms by which genes are regulated may advance the understanding of factors influencing pathogen behavior or cellular physiology. This work finds that expression of a major facilitator superfamily (MFS) transporter, which is governed by a quorum sensing (QS) system, impacts the expression of multiple secreted virulence factors and accounts for a documented QS-dependent antibiotic susceptibility. Although the mechanism underlying this effect is not clear, MFS orthologs with high sequence similarity from S. pneumoniae and S. porcinus were unable to substitute indicating substrate specificity of the GAS MFS gene. These findings demonstrate novel associations between the expression of a transmembrane transporter and virulence factor expression and aminoglycoside transport.

A requirement for Kruppel-Like Factor-4 in the maintenance of endothelial cell quiescence

Mastej, V. — 2022-05-10

Rationale and GoalEndothelial cells (ECs) are quiescent and critical for maintaining homeostatic functions of the mature vascular system, while disruption of quiescence is at the heart of endothelial to mesenchymal transition (EndMT) and tumor angiogenesis. Here, we addressed the hypothesis that KLF4 maintains the EC quiescence. Methods and ResultsIn ECs, KLF4 bound to KLF2, and the KLF4-transctivation domain (TAD) interacted directly with KLF2. KLF4-depletion increased KLF2 expression, accompanied by phosphorylation of SMAD3, increased expression of alpha-smooth muscle actin (SMA), VCAM-1, TGF-{beta}1 and ACE2, but decreased VE-cadherin expression. In the absence of Klf4, Klf2 bound to the Klf2-promoter/enhancer region and autoregulated its own expression. Loss of EC-Klf4 in RosamT/mG::Klf4fl/fl::Cdh5CreERT2 engineered mice, increased Klf2 levels and these cells underwent EndMT. ConclusionIn quiescent ECs, KLF2 and KLF4 partnered to regulate a combinatorial mechanism. The loss of KLF4 disrupted this combinatorial mechanism, thereby upregulating KLF2 as an adaptive response. However, increased KLF2 expression overdrives for the loss of KLF4, giving rise to an EndMT phenotype. Key PointsAdult endothelial cells (ECs) are quiescent in that these cells are arrested at G0-phase of the cell cycle, but mechanisms of EC quiescence are not well understood. The Kruppel-like factors (KLFs) -2 and -4 are transcriptional regulators, highly expressed in quiescent ECs, however, their roles in this process have not been addressed. Elucidation of the mechanisms of KLF function in quiescent ECs should provide clues to the translational discoveries intended for the treatment of EC-dysfunction, such as endothelial to mesenchymal transition (EndMT) associated with several vascular diseases including tumor angiogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=162 HEIGHT=200 SRC="FIGDIR/small/491221v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@8f38d2org.highwire.dtl.DTLVardef@db74b7org.highwire.dtl.DTLVardef@1cbd029org.highwire.dtl.DTLVardef@1cf440d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Extraintestinal roles of intestinal vitamin D receptor in protecting against dysbiosis and tumorigenesis in breast cancer

Zhang, Y.-g. — 2022-05-19

The microbiota play critical roles in regulating the function and health of intestine and extraintestinal organs. A fundamental question is whether there is an intestinal-microbiome-breast axis during the development of breast cancer. If yes, what are the roles of host factors? Vitamin D receptor (VDR) involves host factors and the human microbiome. Vdr gene variation shapes the human microbiome and VDR deficiency leads to dysbiosis. We hypothesized that intestinal VDR protects hosts against tumorigenesis in breast. Reduced VDR mRNA expression was observed in patients with breast cancer. We used a 7,12-dimethylbenzanthracene (DMBA)-induced breast cancer model in intestinal epithelial VDR knockout (VDR{Delta}IEC) mice. We reported that VDR{Delta}IEC mice with dysbiosis are more susceptible to breast cancer induced by DMBA. Intestinal and breast microbiota analysis showed that lacking VDR leads to bacterial profile shift from normal to susceptible carcinogenesis. We found enhanced bacterial staining within breast tumors. At the molecular and cellular levels, we identified the mechanisms by which intestinal epithelial VDR deficiency led to increased gut permeability, disrupted tight junctions, microbial translocation, and enhanced inflammation, thus increasing the tumor size and number in breast. Furthermore, treatment with beneficial bacterial metabolite butyrate or probiotic Lactobacillus plantarum reduced the breast tumors, enhanced the tight junctions, and inhibited inflammation in the VDR{Delta}IEC mice. Gut microbiome contribute to the pathogenesis of diseases, not only in the intestine, but also in the breast. Our study provides new insights into the mechanism by which intestinal VDR dysfunction and gut dysbiosis led to high risk of extraintestinal tumorigenesis. Gut-tumor-microbiome interactions indicate a new target in the prevention and treatment of breast cancer. O_FIG O_LINKSMALLFIG WIDTH=165 HEIGHT=200 SRC="FIGDIR/small/492300v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@7e354eorg.highwire.dtl.DTLVardef@1dd9b50org.highwire.dtl.DTLVardef@1ffe2c5org.highwire.dtl.DTLVardef@810b1a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Dysfunction of transfer RNA modifications in inflammatory bowel disease

Zhang, J. — 2022-05-19

Backgrounds and aimsTransfer RNA (tRNA) is the most extensively modified RNA in cells. Queuosine (Q)-modification is a fundamental process for fidelity and efficiency of translation from RNA to proteins. In eukaryotes, tRNA-Q-modification relies on the intestinal microbial product queuine. However, the roles and potential mechanisms of Q-tRNA modifications in IBD are unknown. MethodsWe explored the Q-tRNA modifications and expression of Q tRNA ribosyltransferase catalytic subunit 1 (QTRT1) in patients with IBD by investigating human biopsies and reanalyzing datasets. We used colitis models, organoids, and cultured cells for loss- and gain-of-function studies to investigate the molecular mechanisms of Q-tRNA modifications in intestinal inflammation. ResultsQTRT1 expression was significantly downregulated in ulcerative colitis and Crohns disease patients. The four Q-tRNA-related tRNA synthetases (asparaginyl-aspartyl-, histidyl-, and tyrosyl-tRNA synthetase) were decreased in IBD patients. This reduction was further confirmed in DSS-induced colitis and IL10-deficient mice. Reduced QTRT1 was significantly correlated with cell proliferation and intestinal junctions, including downregulated {beta}-catenin and Claudin-5 and upregulated Claudin-2. These alterations were confirmed in vitro by deleting QTRT1 from cells. Queuine treatment significantly enhanced cell proliferation and junction functions in cell lines and human colonoids. Queuine treatment also reduced inflammation in epithelial cells. Moreover, altered QTRT1-related metabolites were found in human IBD. ConclusiontRNA modifications play an unexplored novel role in the pathogenesis of intestinal inflammation by altering epithelial proliferation and junctions. Investigations on tRNA modification will uncover novel molecular mechanisms for potential prevention and therapy for IBD.

Long-term functional regeneration of radiation damaged salivary glands through delivery of a neurogenic hydrogel

Li, J. — 2022-05-19

Salivary gland acinar cells are severely depleted after radiotherapy for head and neck cancer, leading to loss of saliva and extensive oro-digestive complications. With no regenerative therapies available, organ dysfunction is irreversible. Here using the adult murine system, we demonstrate radiation-damaged salivary glands can be functionally regenerated via sustained delivery of the neurogenic muscarinic receptor agonist, cevimeline. We show that endogenous gland repair coincides with increased nerve activity and acinar cell division that is limited to the first week post-radiation, with extensive acinar cell degeneration, dysfunction and cholinergic denervation occurring thereafter. However, we discovered that mimicking cholinergic muscarinic input via sustained local delivery of a cevimeline-alginate hydrogel was sufficient to regenerate innervated acini and retain physiological saliva secretion at non-irradiated levels over the long-term (> 3 months). Thus, we reveal a novel regenerative approach for restoring epithelial organ structure and function that has significant implications for human patients. TeaserNovel application of an injectable neurogenic-based hydrogel for restoring the structure and function of radiation-damaged tissue.

Increased GABA transmission to GnRH neurons after intrahippocampal kainic acid injection in mice is sex-specific and associated with estrous cycle disruption

Ingram, R. J. — 2022-05-21

Patients with epilepsy develop reproductive endocrine comorbidities at a rate higher than that of the general population. Clinical studies have identified disrupted luteinizing hormone (LH) release patterns in patients of both sexes, suggesting potential epilepsy-associated changes in hypothalamic gonadotropin-releasing hormone (GnRH) neuron function. In previous work, we found that GnRH neuron firing is increased in diestrous females and males in the intrahippocampal kainic acid (IHKA) mouse model of temporal lobe epilepsy. Notably, GABAA receptor activation is depolarizing in adult GnRH neurons. Therefore, here we tested the hypothesis that increased GnRH neuron firing in IHKA mice is associated with increased GABAergic drive to GnRH neurons. When ionotropic glutamate receptors (iGluRs) were blocked to isolate GABAergic postsynaptic currents (PSCs), no differences in PSC frequency were seen between GnRH neurons from control and IHKA diestrous females. In the absence of iGluR blockade, however, GABA PSC frequency was increased in GnRH neurons from IHKA females with disrupted estrous cycles, but not saline-injected controls nor IHKA females without estrous cycle disruption. GABA PSC amplitude was also increased in IHKA females with disrupted estrous cycles. These findings suggest the presence of an iGluR-dependent increase in feed-forward GABAergic transmission to GnRH neurons specific to IHKA females with comorbid cycle disruption. In males, GABA PSC frequency and amplitude were unchanged but PSC duration was reduced. Together, these findings suggest that increased GABA transmission helps drive elevated firing in IHKA females on diestrus and indicate the presence of a sex-specific hypothalamic mechanism underlying reproductive endocrine dysfunction in IHKA mice. HIGHLIGHTSO_LIIncreased GABA transmission to GnRH neurons in IHKA mouse model of epilepsy C_LIO_LIIncreased GABA transmission is dependent on upstream glutamate signaling C_LIO_LIIncreased GABA transmission only seen in females with disrupted estrous cycles C_LIO_LIPotential sex-specific mechanism for reproductive endocrine dysfunction in epilepsy C_LI

An Igh novel enhancer modulates antigen receptor diversity by determining locus conformation

Bhat, K. H. — 2022-05-24

The Igh locus is organized into a developmentally regulated topologically associated domain (TAD) that is divided into subTADs. Here we identify a series of novel enhancers (NEs) that collaborate to configure the locus, determine transcriptional potential in over a hundred functional VH genes and their usage in V(D)J recombination. NE1 engages in a network of long-range interactions that interconnect the subTADs and the recombination center at the DHJH gene cluster. Deletion of NE1 alters discrete chromatin loops, higher order locus conformation, locus-wide VH gene transcription and regional V gene utilization that is linked to a greatly reduced splenic B1 B cell compartment. NE1 blocks long-range loop extrusion that in turn contributes to locus contraction and determines the proximity of distant VH genes to the recombination center. NE1 is a critical architectural element that coordinates chromatin conformational states that favor VH gene transcription or V(D)J rearrangement.

Gut Microbes and the Liver Circadian Clock Partition Glucose and Lipid Metabolism

Frazier, K. — 2022-05-25

Circadian rhythms govern glucose homeostasis, and their dysregulation leads to complex metabolic diseases. Gut microbes also exhibit diurnal rhythms that influence host circadian networks and metabolic processes, yet underlying mechanisms remain elusive. Here, we show hierarchical, bi-directional communication between the liver circadian clock, gut microbes, and glucose homeostasis in mice. The liver clock, but not the forebrain clock, requires gut microbes to drive glucose clearance and gluconeogenesis. Liver clock dysfunctionality expands proportions and abundances of oscillating microbial features by two-fold relative to controls. The liver clock is the primary driver of differential and rhythmic hepatic expression of glucose and fatty acid metabolic pathways. Absent the liver clock, gut microbes provide secondary cues that dampen these rhythms, resulting in reduced utilization of lipids as fuel relative to carbohydrates. Together, the liver clock transduces signals from gut microbes necessary to regulate glucose and lipid metabolism and meet energy demands over 24 hours. HighlightsThe liver circadian clock is autonomous from the central clock in metabolic regulation Liver clock and gut microbes interact to direct hepatic glucose and lipid metabolism Reciprocating host-microbe interactions drive rhythmic hepatic transcription Perturbed liver Bmal1 results in chaotic downstream oscillators and metabolism

G protein βγ subunits bind to and inhibit the function of multiple Qa- and Qb,c-SNARE isoforms.

Zurawski, Z. — 2022-05-30

While the ability of G protein {beta}{gamma} subunits (G{beta}{gamma}) to bind to and functionally inhibit the neuronal SNARE proteins Stx1A, SNAP25, and synaptobrevin in the presence of the calcium sensor synaptotagmin I is well documented, these three SNARE proteins, which form the core SNARE complex for synchronous evoked release in neurons, are but a subset of the larger family of SNARE proteins, which participate in many other exocytic processes within the cell and in other populations of secretory cells throughout the body, from which the release of neurotransmitters, hormones, and other factors is regulated by Gi/o-coupled GPCRs. The ability of G{beta}{gamma} to regulate these processes is unknown. To investigate the feasibility of this mechanism to inhibit SNARE function more broadly, we utilized a series of biochemical assays of binding and function with four Qa-SNAREs (Stx1A, Stx2, Stx3, and Stx4) and four Qb,c-SNAREs (SNAP25, SNAP23, SNAP29, and SNAP47) in tandem with the R-SNARE synaptobrevin, synaptotagmin I, and G{beta}{gamma}. G{beta}{gamma} was found to bind to multiple Qa-SNARE isoforms as well as SNAP23, and inhibit the lipid mixing function of these SNAREs, as well as SNAP29. Together, this data suggests a more broad role for the G{beta}{gamma}-SNARE pathway in the regulation of exocytosis beyond cells that express Stx1A or SNAP25.

Food deprivation modulates heart rate, motor neuron, and locomotion responses to acute administration of d-amphetamine in zebrafish larvae

Bansal, P. — 2022-06-01

Psychostimulant drugs are so named because they alter the cardiac, brain and behavioral responses in humans and other animals. Acute food deprivation or chronic food restriction potentiates the stimulatory effects of abused drugs and increases the propensity for relapse to drug seeking in drug-experienced animals. The mechanisms by which hunger affects cardiac and behavioral activities are only beginning to be elucidated. Moreover, changes in motor neuron activities at the single neuron level induced by the stimulants, and their modulation by hunger, remain unknown. Here we investigated how the state of hunger affects responses to d-amphetamine by measuring locomotion, cardiac output, and individual motor neuron activity in zebrafish larvae. We used wild-type larval zebrafish to record behavioral and cardiac responses and the larvae of mnx1:GCaMP transgenic zebrafish to record motor neuron responses. Acute administration of d-amphetamine in sated larvae did not induce a significant change in the motor responses (swimming distances, tail activity), heart rate, or motor neuron firing frequency to the stimulant. However, food deprivation enhanced amphetamine-evoked responses significantly. The results extend the finding that signals arising from food deprivation are a key potentiator of the drug responses induced by d-amphetamine to the zebrafish model. The larval zebrafish is an ideal model to further elucidate this interaction and identify key neuronal substrates that may increase vulnerability to drug reinforcement, drug-seeking and relapse.

Age exacerbates SARS-CoV-2-induced blood-brain barrier leakage and neuropsychiatric dysfunction

Niladhuri, S. B. — 2022-06-03

Respiratory infection with SARS-CoV-2 causes systemic vascular inflammation and cognitive impairment. We sought to identify the underlying mechanisms mediating vascular dysfunction and inflammation following mild respiratory SARS-CoV-2 infection. To this end, we conduced unbiased transcriptional analysis to identify brain endothelial cell signaling pathways dysregulated by SARS-CoV-2 in vivo. This analysis revealed significant suppression of Wnt/{beta}-catenin signaling, a critical regulator of blood brain barrier integrity. We therefore hypothesized that enhancing cerebrovascular Wnt/{beta}-catenin activity would offer protection against BBB permeability, neuroinflammation, and neurological signs in acute infection. Indeed, we found that delivery of cerebrovascular-targeted, engineered Wnt7a ligands protected blood brain barrier integrity, reduced T cell infiltration of the brain, and reduced microglial activation in SARS-CoV-2 infection. Importantly, this therapeutic strategy also mitigated SARS-CoV-2 induced deficits in the novel object recognition assay for learning and memory and the pole descent task for bradykinesia. These observations suggest that enhancement of Wnt/{beta}-catenin signaling or its downstream effectors could be potential interventional strategies for restoring cognitive health following acute viral infections.

Identification of structural features of selected flavonoids responsible for neuroprotection using a Drosophila model of Parkinson's disease

Maitra, U. — 2022-06-04

Nature-derived bioactive compounds have emerged as promising candidates for the prevention and treatment of diverse chronic illnesses, including neurodegenerative diseases. However, the exact molecular mechanisms underlying their neuroprotective effects remain unclear. Most studies focus solely on the antioxidant activities of natural products which translate to poor outcome in clinical trials. Current therapies against neurodegeneration only provide symptomatic relief thereby underscoring the need for novel strategies to combat disease onset and progression. We have employed an environmental toxin-induced Drosophila Parkinsons disease (PD) model as an inexpensive in vivo screening platform to explore neuroprotective potential of selected dietary flavonoids. We have identified a specific group of flavonoids known as flavones displaying protection against paraquat (PQ)-induced neurodegenerative phenotypes, involving reduced survival, mobility defects and enhanced oxidative stress. Interestingly, the other groups of investigated flavonoids, namely, the flavonones and flavonols failed to provide protection indicating a requirement of specific structural features that confer protection against PQ-mediated neurotoxicity in Drosophila. Based on our screen, the neuroprotective flavones lack a functional group substitution at the C3 and contain ,{beta}-unsaturated carbonyl group. Furthermore, flavones-mediated neuroprotection is not solely dependent on antioxidant properties but also involves regulation of neuroinflammatory responses. Our data identify specific structural features of selected flavonoids that provide neuroprotection against environmental toxin-induced PD pathogenesis that can be explored for novel therapeutic interventions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/494711v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@130933aorg.highwire.dtl.DTLVardef@11e959org.highwire.dtl.DTLVardef@1eef5fforg.highwire.dtl.DTLVardef@1585a2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hepatocyte PPARγ contributes to the progression of non-alcoholic steatohepatitis in male and female obese mice.

Lee, S. M. — 2022-06-08

Background & AimsNon-alcoholic steatohepatitis (NASH) is associated with obesity and increased expression of hepatic peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) in humans. Although we previously showed that the expression of PPAR{gamma} in hepatocytes contributes to the development NASH in lean mice, the relevance of hepatocyte PPAR{gamma} in the development of NASH associated with obesity is still poorly understood. MethodsHepatocyte PPAR{gamma} was knocked out (Pparg{Delta}Hep) after the development of high-fat diet-induced obesity in male and female mice and before NASH was induced with a high fat, cholesterol and fructose (HFCF) diet. We assessed the effect of the diets and Pparg{Delta}Hep on body composition and glucose homeostasis, as well as on the liver pathology, gene expression, and metabolome. In addition, liver biopsies from a cohort of 102 bariatric surgery patients were assessed for liver histology and gene expression. ResultsPPAR{gamma} expression, specifically PPAR{gamma}2, is mostly derived from hepatocytes and increased by high fat diets. Pparg{Delta}Hep reduced HFCF-induced NASH progression without altering steatosis. Interestingly, Pparg{Delta}Hep reduced the expression of key genes involved in hepatic fibrosis in HFCF-fed male and female mice, and collagen- stained fibrotic area in the liver of HFCF-fed male mice. In addition, transcriptomic and metabolomic data suggested that HFCF-diet regulated hepatic amino acid metabolism in a hepatocyte PPAR{gamma}-dependent manner. Specifically, Pparg{Delta}Hep increased betaine-homocysteine methyltransferase expression and reduced homocysteine levels in HFCF- fed male mice. In a cohort of 102 bariatric surgery patients, 16 cases of NASH were associated with increased insulin resistance and hepatic PPAR{gamma} expression. ConclusionsHepatocyte PPAR{gamma} expression associated with obesity could regulate methionine metabolism and the progression of fibrosis in NASH.

A kinesin mediates VEGFR2 recycling and regulates VE-cadherin phosphorylation, essential for vascular permeability

Cho, H.-D. — 2022-06-06

Excessive vascular endothelial growth factor-A (VEGF-A) signaling induces vascular leakage and angiogenesis in diseases. VEGFR2 trafficking to the cell surface, mediated by kinesin-3 family protein KIF13B, is essential to respond to VEGF-A in inducing angiogenesis. However, the precise mechanism of how KIF13B regulates VEGF-induced signaling and endothelial permeability is unknown. Here we show that KIF13B-mediated recycling of internalized VEGFR2 through Rab11-positive recycling vesicle regulates VE-cadherin phosphorylation and endothelial permeability. Phosphorylated VEGFR2 at the cell-cell junction was internalized and associated with KIF13B in Rab5-positive early endosomes. KIF13B mediated VEGFR2 recycling through Rab11-positive recycling vesicle, and inhibition of this recycling attenuated phosphorylation of VEGFR2 at Y951, Src, and VE-cadherin at Y685, which are necessary for endothelial permeability. Failure of VEGFR2 trafficking to the cell surface induced accumulation and degradation of VEGFR2 in lysosomes. Furthermore, in the animal model of wet age-related macular degeneration (AMD), inhibition of KIF13B-mediated VEGFR2 trafficking also mitigated vascular leakage. Thus, the present results identify the fundamental role of VEGFR2 recycling to the cell surface in mediating vascular permeability, suggesting a promising strategy for mitigating vascular leakage associated with inflammatory diseases.

Role of TM3 in claudin-15 strand flexibility: a molecular dynamics study

Fuladi, S. — 2022-06-09

Claudins are cell-cell adhesion proteins within tight junctions that connect epithelial cells together. Claudins polymerize into a network of strand-like structures within the membrane of adjoining cells and create ion channels that control paracellular permeability to water and small molecules. Tight junction morphology and barrier function is tissue specific and regulated by claudin subtypes. Here, we present a molecular dynamics study of claudin-15 strands within lipid membranes and the role of a single-point mutation (A134P) on the third transmembrane helix (TM3) of claudin-15 in determining the morphology of the strand. Our results indicate that the A134P mutation significantly affects the lateral flexibility of the strands, increasing the persistence length of claudin-15 strands by a factor of three. Analyses of claudin-claudin contact in our {micro}second-long trajectories show that the mutation does not alter the intermolecular contacts (interfaces) between claudins. However, the dynamics and frequency of interfacial contacts are significantly affected. The A134P mutation introduces a kink in TM3 of claudin-15 similar to the one observed in claudin-3 crystal structure. The kink on TM3 skews the rotational flexibility of the claudins in the strands and limits their fluctuation in one direction. This asymmetric movement in the context of the double rows reduces the lateral flexibility of the strand and leads to higher persistence lengths of the mutant.

SpiDec: Computing Binodals and Interfacial Tension of Biomolecular Condensates From Simulations of Spinodal Decomposition

Mazarakos, K. — 2022-06-17

Phase separation of intrinsically disordered proteins (IDPs) is a phenomenon associated with many essential cellular processes, but a robust method to compute the binodal from molecular dynamics simulations of IDPs modeled at the all-atom level in explicit solvent is still elusive, due to the difficulty in preparing a suitable initial dense configuration and in achieving phase equilibration. Here we present SpiDec as such a method, based on spontaneous phase separation via spinodal decomposition that produces a dense slab when the system is initiated at a homogeneous, low density. After illustrating the method on four model systems, we apply SpiDec to a tetrapeptide modeled at the all-atom level and solvated in TIP3P water. The concentrations in the dense and dilute phases agree qualitatively with experimental results and point to binodals as a sensitive property for force-field parameterization. SpiDec may prove useful for the accurate determination of the phase equilibrium of IDPs. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/496322v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@118b545org.highwire.dtl.DTLVardef@124e03eorg.highwire.dtl.DTLVardef@1fe3c5forg.highwire.dtl.DTLVardef@2dbc77_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pre-existing immunity modulates responses to mRNA boosters

Dangi, T. — 2022-06-28

mRNA vaccines have shown high efficacy in preventing severe COVID-19, but breakthrough infections, emerging variants and waning antibody levels have warranted the use of boosters. Although mRNA boosters have been widely implemented, the extent to which pre-existing immunity influences the efficacy of boosters remains unclear. In a cohort of individuals primed with the mRNA-1273 or BNT162b2 vaccines, we observed that lower antibody levels before boost were associated with higher fold-increase in antibody levels after boost, suggesting that pre-existing antibody modulates the boosting capacity of mRNA vaccines. Mechanistic studies in mice show that pre-existing antibodies significantly limit antigen expression and priming of B cell responses after mRNA vaccination. Furthermore, we demonstrate that the relative superiority of an updated Omicron vaccine over the original vaccine is critically dependent on the serostatus of the host. These data demonstrate that pre-existing immunity dictates responses to mRNA vaccination, elucidating specific circumstances when updated SARS-CoV-2 vaccines confer superior protection to original vaccines.

The Entwined African and Asian Genetic Roots of the Medieval Peoples of the Swahili Coast

Brielle, E. S. — 2022-07-11

The peoples of the Swahili coast of eastern Africa established a literate urban culture by the second millennium CE. They traded across eastern Africa and the Indian Ocean and were among the first sub-Saharan practitioners of Islam. An open question has been the extent to which these early interactions between Africans and non-Africans were accompanied by genetic admixture. We report genome-wide ancient DNA from 80 individuals in five medieval and early modern (1300-1800 CE) coastal towns, as well as people from an inland town postdating 1650 CE. Over half of the ancestry of most coastal individuals came from African ancestors; these African ancestors were primarily female. A slightly smaller proportion of ancestry was from Asia. This Asian component was approximately eighty to ninety percent from Near Eastern males and ten to twenty percent from Indian females. Peoples of African and Asian origins began to mix by around 1000 CE, a time when archaeological evidence documents changes on the coast that are often interpreted as marking the large-scale adoption of Islam. Before roughly 1500 CE, the Near Eastern ancestry detected in the individuals was mainly Persian-related, consistent with the narrative of the Kilwa Chronicle, the oldest history told by the Swahili themselves. After this time, the sources of Near Eastern ancestry became increasingly Arabian, consistent with the archaeological and historical evidence of growing interactions between the Swahili coast and parts of southern Arabia. Subsequent interactions of Swahili coast peoples with other Asian and African groups further changed the ancestry of present-day peoples relative to the ancient individuals we sequenced, highlighting how Swahili genetic legacies can be more clearly understood with ancient DNA.

Automated container-less cell processing method for single-cell proteomics

Matsumoto, C. — 2022-07-27

Single-cell genomics and transcriptomics studies enabled us to characterize cell heterogeneity in various tissues, which helped us to better understand the biological system and disease progression. Single-cell proteomics, which directly measures the protein expression level, has the potential to further enhance our knowledge by providing not only a more direct measurement but also crucial information cannot be captured by genomics or transcriptomics study, such as protein activation states and post-translation modification events. One of the main challenges of single-cell proteomics is the large sample loss during sample preparation, which is largely unavoidable using standard proteomics protocols. Protein and peptide loss to the container surface is a well-known phenomenon but often overlooked in larger-scale (>1 {micro}g) proteomics experiments. When it comes to single-cell proteomics with only picograms of protein samples, this loss becomes non-negligible and often dictates the outcomes of the experiment. More importantly, sample processing through multiple pipette tips and containers often introduces random errors, which undermine the ability to detect true heterogenous cellular events. To solve these problems and further improve the throughput and reproducibility of the single-cell proteomics experiments, we developed an automated container-less cell processing platform, utilizing acoustic levitation to process cell samples in the air. Our platform automatically performs cell lysis, protein reduction, alkylation, digestion, and peptide labeling in the air, without any sample transfer step or container. The digested and labeled peptides are then directly injected into the capillary LC-MS/MS system for analysis, eliminating manual steps and conserving most of the sample materials for proteomics analysis. Our initial test shows at least 30% improvement in peptide signals over conventional methods. This process can be performed in parallel to further improve sample processing throughput.

ROP16-mediated activation of STAT6 facilitates encystment of type III Toxoplasma gondii in neurons

Kochanowsky, J. A. — 2022-07-31

Toxoplasma gondii establishes a long-lived latent infection in the central nervous system (CNS) of its hosts. Reactivation in immunocompromised individuals can lead to life threatening disease. Latent infection is driven by the ability of the parasite to convert from the acute-stage tachyzoite to the latent-stage bradyzoite which resides in long-lived intracellular cysts. While much work has focused on the parasitic factors that drive cyst development, the host factors that influence encystment are not well defined. Here we show that a polymorphic secreted parasite kinase (ROP16), that phosphorylates host cell proteins, mediates efficient encystment of T. gondii in stress-induced models of encystment and primary neuronal cell cultures (PNCs) in a strain-specific manner. Using short-hairpin RNA (shRNA) knockdowns in human foreskin fibroblasts (HFFs) and PNCs from transgenic mice, we determined that ROP16s cyst enhancing abilities are mediated by phosphorylation of the host cell transcription factor STAT6. To test the role of STAT6 in vivo, we infected STAT6KO mice, finding that, compared to infected wild-type mice, infected STAT6KO mice have a decrease in cyst burden, but not overall parasite burden or dissemination to the CNS. Finally, we found a similar ROP16-dependent encystment defect in human pluripotent stem cell-derived neurons. Together, these findings identify a host cell factor (STAT6) that T. gondii manipulates in a strain-specific manner to generate a favorable encystment environment.

Pink1-mediated mitophagy in the endothelium releases proteins encoded by mitochondrial DNA and activates neutrophil responses

Gajwani, P. — 2022-08-07

Given their ancient evolutionary origins, eukaryotic mitochondria possess multiple vestiges of their prokaryotic ancestors. One such factor is the N-terminal formylation of proteins encoded by mitochondrial DNA. N-formylated proteins are also released by bacteria and trigger activation of immune cells such as neutrophils. Growing evidence indicate that circulating levels of mitochondrial formyl proteins are elevated in the serum of patients with excessive inflammatory responses and trigger neutrophil activation like their bacterial counterparts. However, the cellular source of these proteins, and the mechanism by which they are released into the circulation is not known. In this study, we have identified vascular endothelial cells as a source of mitophagy induced release of formyl proteins in response to inflammatory mediators in vitro. Mechanistically, endothelial mitophagy required activation of the Pink1 pathway. Using liposomal delivery of sgRNA targeting Pink1 in mice expressing endothelial-specific Cas9, we developed a mouse model in which Pink1 is specifically depleted in the endothelium. Deletion of endothelial Pink1 was remarkably protective in endotoxin-induced lung inflammation, resulting in reduced neutrophil infiltration and significantly reduced death in mice. We thus propose that endothelial cells upregulate pro-inflammatory mitophagy in response to inflammation, leading to release of mitochondrial formyl peptides and detrimental neutrophil recruitment into the lung.

STAT3 signaling in B cells controls germinal center zone organization and recycling

Fike, A. J. — 2022-08-15

Germinal centers (GCs), sites of antibody affinity maturation, are organized into dark (DZ) and light (LZ) zones. Here, we uncovered a B cell intrinsic role for STAT3 in GC DZ and LZ organization. Altered zonal organization of STAT3-deficient GCs dampened GC output of long-lived plasma cells (LL-PCs) but increased memory B cells (MBCs). Tfh-GC B cell interaction drive STAT3 tyrosine 705 and serine 727 phosphorylation in LZ B cells, facilitating their recycling into the DZ. An inducible system confirmed STAT3 is not involved in initiating or maintaining the GC but sustains GC zonal organization by regulating GC B cell recycling. RNAseq and ChIPseq analysis identified genes regulated by STAT3 that are critical for LZ cell recycling and transiting through the DZ proliferation and differentiation phases of the DZ. Thus, STAT3 signaling in B cells controls GC zone organization and recycling, and GC egress of LL-PCs, but negatively regulates MBC output. SummaryFike et al. describe a previously unrecognized mechanism by which B cell intrinsic STAT3 signaling controls the germinal center (GC) dark and light zone organization, GC B cell recycling, and GC output of long-lived plasma cells but negatively regulates memory B cells.

Direct conversion of somatic cells into insulin-producing-cells by user-defined multiplex-epigenetic-engineering vector (MEEV-b)

Naqvi, R. — 2022-08-13

We demonstrate here a single-step and user-friendly approach to generate insulin producing cells by gRNA driven specific-activation of PDX1, NKX6.1, MAFA, Insulin and Glut2 genes in somatic cells via multiplex-epigenetic-engineering-vector (MEEV-{beta}) containing dCas9.P300core developed by us. Sorted Glut2+ cells could secrete insulin in response to glucose challenge and showed expression of {beta}-cell specific transcription factors: NKX2.2, and aforementioned genes. Expression of Cav1.3, GSK3{beta},, KJNC11, and SLC30A8 genes substantiated the functional insulin secreting machinery genes in these Glut2+ cells. Also, absence of ARX and GCG expression in these cells highlighted the specificity of the conversion.

Membrane Tethering of SepF, a Membrane Anchor for the Mycobacterium tuberculosis Z-ring

Dey, S. — 2022-08-26

Bacterial cell division begins with the formation of the Z-ring via polymerization of FtsZ and the localization of Z-ring beneath the inner membrane through membrane anchors. In Mycobacterium tuberculosis (Mtb), SepF is one such membrane anchor, but our understanding of the underlying mechanism is very limited. Here we used molecular dynamics simulations to characterize how SepF itself, a water-soluble protein, tethers to acidic membranes that mimic the Mtb inner membrane. In addition to an amphipathic helix (residues 1-12) at the N-terminus, membrane binding also occurs through two stretches of positively charged residues (Arg27-Arg 37 and Arg95-Arg107) in the long linker preceding the FtsZ-binding core domain (residues 128-218). The additional interactions via the disordered linker stabilize the membrane tethering of SepF, and keep the core domain of SepF and hence the attached Z-ring close to the membrane. The resulting membrane proximity of the Z-ring in turn enables its interactions with and thus recruitment of two membrane proteins, FtsW and CrgA, at the late stage of cell division.

An Arg/Ala-Rich Helix in the N-Terminal Region of M. tuberculosis FtsQ Anchors FtsZ to Membranes

Escobar, C. A. — 2022-09-03

Mycobacteria tuberculosis (Mtb) inflicts a quarter of the worldwide population. Most drugs for treating tuberculosis target cell growth and division. With rising drug resistance, it becomes ever more urgent to better understand Mtb cell division. This process begins with the formation of the Z-ring via polymerization of FtsZ and anchoring of the Z-ring to the inner membrane. Here, by combining solution and solid-state NMR spectroscopy with molecular dynamics simulations and other techniques, we show that the transmembrane protein FtsQ is a membrane anchor of the Mtb Z-ring. In the otherwise disordered N-terminal cytoplasmic region of FtsQ (residues 1-99), a 29-residue, Arg/Ala-rich -helix is formed that interacts with upstream acidic residues in solution and with acidic lipids at the membrane surface. The same helix also binds to the GTPase domain of FtsZ, with enormous implications for drug binding and Z-ring formation including its curvature.

The cytidine deaminase APOBEC3G drives cancer mutagenesis and clonal evolution in bladder cancer

Liu, W. — 2022-09-05

Mutagenic processes leave distinct signatures in cancer genomes. The mutational signatures attributed to APOBEC3 cytidine deaminases are pervasive in human cancers. However, data linking individual APOBEC3 proteins to cancer mutagenesis in vivo are limited. Here, we show that transgenic expression of human APOBEC3G promotes mutagenesis, genomic instability, and kataegis, leading to shorter survival in a murine bladder cancer model. Acting as mutagenic fuel, APOBEC3G increases the clonal diversity of bladder cancers, driving divergent cancer evolution. We characterize the single base substitution signature induced by APOBEC3G in vivo, showing the induction of a mutational signature different from that caused by APOBEC3A and APOBEC3B. Analysis of thousands of human cancers reveals the contribution of APOBEC3G to the mutational profiles of multiple cancer types, including bladder cancer. Our findings define the role of APOBEC3G in cancer mutagenesis and clonal heterogeneity. These results potentially inform future therapeutic efforts that restrict tumor evolution.

Estrogen signaling in the dorsal raphe regulates binge-like drinking in mice

Torres Irizarry, V. C. — 2022-09-06

The ovarian hormone estrogens promote binge alcohol drinking and contribute to sex differences in alcohol use disorder. However, the mechanisms for estrogen-induced binge drinking are largely unknown. This study aims to test if estrogens act on 5-hydroxytryptamine neurons in the dorsal raphe nucleus (5-HTDRN) to promote binge drinking. We used the drinking in the dark (DID) behavioral test in mice to mimic binge drinking in humans. We found that female mice drank more alcohol than male mice in chronic DID tests. This sex difference was associated with distinct alterations in mRNA expression of estrogen receptor (ER) and 5-HT-related genes in the DRN, suggesting a potential role of estrogen/ERs/5-HT signaling in binge alcohol drinking. In supporting this view, 5-HTDRN neurons from naive male mice had lower baseline neuronal firing activity but higher sensitivity to alcohol-induced excitation compared to 5-HTDRN neurons from naive female mice. Notably, this higher sensitivity was blunted by 17{beta}-estradiol treatment in males, indicating an estrogen-dependent mechanism. We further showed that both ER and ER{beta} are expressed in 5-HTDRN neurons, whereas ER agonist propyl pyrazole triol (PPT) depolarizes 5-HTDRN neurons and ER{beta} agonist diarylpropionitrile (DPN) hyperpolarizes 5-HTDRN neurons. Notably, both PPT and DPN treatments blocked the stimulatory effects of alcohol on 5-HTDRN neurons in males, despite the fact that they have antagonistic effects on the activity dynamics of 5-HTDRN neurons. These results suggest that ERs inhibitory effects on ethanol-induced burst firing of 5-HTDRN neurons may contribute to higher levels of binge drinking in females. Consistently, chemogenetic activation of ER- or ER{beta}-expressing neurons in the DRN reduced binge alcohol drinking. These results support a model in which estrogens act on ER/{beta} to prevent alcohol-induced activation of 5-HTDRN neurons, which in return leads to higher binge alcohol drinking.

Topical GZ21T inhibits the growth of actinic keratoses in a UVB induced model of skin carcinogenesis

Bordeaux, Z. A. — 2022-09-07

Actinic keratoses (AKs) are premalignant intraepidermal neoplasms that occur as a result of cumulative sun damage. AKs commonly relapse, and up to 16% undergo malignant transformation into cutaneous squamous cell carcinoma (cSCC). There is a need for novel therapies that reduce the quantity and surface area of AKs as well as prevent malignant transformation to cSCCs. We recently showed that GZ17-6.02, an anti-cancer agent composed of curcumin, haramine, and isovanillin, inhibited the growth of H297.T cells. The present study evaluated the efficacy of a novel topical formulation of GZ17-6.02, known as GZ21T, in a murine model of AK generated by exposing SKH1 mice to ultraviolet irradiation. Treatment of mice with topical GZ21T inhibited the growth of AKs by decreasing both lesion count (p=.028) and surface area occupied by tumor (p=.026). GZ21T also suppressed the progression of AKs to cSCC by decreasing the count (p=.047) and surface area (p=.049) of lesions more likely to represent cSCC. RNA sequencing and proteomic analyses revealed that GZ21T suppressed several pathways, including MAPK (p=.026), Pi3K-Akt (p=.028), HIF-1 (p=.030), Wnt (p=.031), insulin (p=.011), and ErbB (p=.006) signaling. GZ21T also upregulated the autophagy-promoting protein AMPK, while suppressing proteins such as PD-L1, glutaminase, pAkt1 S473, and eEF2K. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/506864v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1254e6corg.highwire.dtl.DTLVardef@3fd190org.highwire.dtl.DTLVardef@1d75eb5org.highwire.dtl.DTLVardef@8afc97_HPS_FORMAT_FIGEXP M_FIG C_FIG

Insights into the molecular mechanism of translation inhibition by the ribosome-targeting antibiotic thermorubin.

Paranjpe, M. N. — 2022-09-16

Thermorubin (THR) is an aromatic anthracenopyranone antibiotic active against both Gram-positive and Gram-negative bacteria. It is known to bind to the 70S ribosome at the intersubunit bridge B2a and was thought to inhibit factor-dependent initiation of translation and obstruct the accommodation of tRNAs into the A site. Here, we show that thermorubin causes ribosomes to stall in vivo and in vitro at internal and termination codons, thereby allowing the ribosome to initiate protein synthesis and translate at least a few codons before stalling. Our biochemical data show that THR affects multiple steps of translation elongation with a significant impact on the binding stability of the tRNA in the A site, explaining premature cessation of translation. Our high-resolution crystal and cryo-EM structures of the 70S-THR complex show that THR can co-exist with P- and A-site tRNAs, explaining how ribosomes can elongate in the presence of the drug. Remarkable is the ability of THR to arrest ribosomes at the stop codons. Our data suggest that by causing structural re-arrangements in the decoding center, THR interferes with the accommodation of tRNAs or release factors into the ribosomal A site. HIGHLIGHTSO_LIThermorubin is a potent inhibitor of protein synthesis both in vivo and in vitro; C_LIO_LIThermorubin does not prevent the binding of P- and A-site tRNAs; C_LIO_LIThermorubin affects multiple steps of translation elongation with a major impact on binding stability of the A-site tRNA; C_LIO_LIThermorubin can act as an inhibitor of translation termination on some ORFs. C_LI

Structure-Based Pathogenicity Relationship Identifier (SPRI): A Novel Structure-Guided Method to Evaluate Pathological Effect of Missense Mutations

Wang, B. — 2022-09-28

We report the Structure-based Pathogenicity Relationship Identifier (SPRI), a novel computational tool for accurate evaluation of pathological effects of missense single mutations and prediction of higher-order spatially organized units of mutational clusters. SPRI can effectively extract properties determining pathogenicity encoded in protein structures, and can identify deleterious missense mutations of germ line origin associated with Mendelian diseases, as well as mutations of somatic origin associated with cancer drivers. It compares favorably to other methods in predicting deleterious mutations. Furthermore, SPRI can discover spatially organized pathogenic higher-order spatial clusters (patHOS) of deleterious mutations, including those of low recurrence, and can be used for discovery of candidate cancer driver genes and driver mutations. We further demonstrate that SPRI can take advantage of AlphaFold2 predicted structures and can be deployed for saturation mutation analysis of the whole human proteome.

Commensal-specific CD4 T cells promote inflammation in the central nervous system via molecular mimicry

White, Z. — 2022-09-28

Commensal bacteria are critical regulators of both tissue homeostasis and the development and exacerbation of autoimmunity. However, it remains unclear how the intestinal microbiota contributes to inflammation in tissues such as the central nervous system (CNS) where these microbes are typically absent and whether T cell receptor (TCR) specificity for commensal-derived antigens is important to the development of tissue inflammation-related outcomes. Here, we found that ileum- and cecum-colonizing segmented filamentous bacteria (SFB)-specific T cells (clone TCR7B8) can infiltrate the CNS wherein they can be reactivated and produce high levels of inflammatory cytokines including IFN{gamma}, IL-17A, TNF, and GM-CSF in the absence of regulatory T cells. In contrast, other SFB-specific T cells (clone TCR1A2) recognizing an epitope in which 8/9 amino acids overlap with those recognized by TCR7B8 failed to induce such neuroinflammation. Despite their similar SFB-derived peptide antigen targets, TCR7B8 was found to recognize peptides derived from host proteins including receptor tyrosine-protein kinase ErbB2, trophinin 1, and anaphase-promoting complex subunit 2 in vitro, whereas TCR1A2 did not, indicating that TCR7B8 induces CNS inflammation via molecular mimicry. Immune checkpoint blockade accelerated TCR7B8-mediated CNS inflammation, suggesting a potential cause of immune-related adverse events induced in cancer patients undergoing such treatment. Together, our findings reveal a potential mechanism whereby gut commensal-specific T cells are dysregulated and contribute to extraintestinal inflammation.

Emulating clinical pressure waveforms in cellculture using an Arduino-controlled 3D-printed platform for 96-well plates

Szmelter, A. H. — 2022-10-03

High blood pressure is the primary risk factor for heart disease, the leading cause of death globally. Despite this, current methods to replicate physiological pressures in-vitro remain limited in sophistication and throughput. Single-chamber exposure systems allow for only one pressure condition to be studied at a time and the application of dynamic pressure waveforms is currently limited to simple sine, triangular, or square waves. Here, we introduce a high-throughput hydrostatic pressure exposure system for 96-well plates. The platform can deliver a fully-customizable pressure waveform to each column of the plate, for a total of 12 simultaneous conditions. Using clinical waveform data, we are able to replicate real patients blood pressures as well as other medically-relevant pressures within the body and have assembled a small patient-derived waveform library of some key physiological locations. As a proof of concept, human umbilical vein endothelial cells (HUVECs) survived and proliferated under pressure for 3 days under a wide range of static and dynamic blood pressures ranging from 10 mm Hg to 400 mm Hg. Interestingly, pathologic and supraphysiologic pressure exposures did not inhibit cell proliferation. By integrating with, rather than replacing, ubiquitous lab cultureware it is our hope that this device will facilitate the incorporation of hydrostatic pressure into standard cell culture practice.

Therapeutic depletion of CD8+ T-cells prevents myelin pathology in Globoid Cell Leukodystrophy

Sutter, P. A. — 2022-10-03

Globoid cell leukodystrophy (GLD) or Krabbes disease is a fatal genetic demyelinating disease of the central nervous system caused by loss-of-function mutations in the galactosylceramidase (galc) gene. While the metabolic basis for disease is known, the understanding of how this results in neuropathology is not well understood. Herein we report that the rapid and protracted elevation of CD8+ cytotoxic T lymphocytes occurs coincident with clinical disease in a mouse model of GLD. Administration of a function blocking antibody against CD8 effectively prevented disease onset, reduced morbidity and mortality and prevented CNS demyelination in mice. These data indicate that subsequent to the genetic cause of disease, neuropathology is driven by pathogenic CD8+ T cells, thus offering novel therapeutic potential for treatment of GLD. One-Sentence SummaryCD8 T-cells mediate demyelination and neuroinflammation in a genetic white matter disease.

Natural selection and neutral mutations through the lens of viruses

Chen, J.-M. — 2022-10-10

The Neutral Theory and the Modern Synthesis, a modified version of Darwins theory, have been arguing for decades about the influence of natural selection on molecular evolution1-10. Here we elucidate through the lens of viruses that a frequently used method11-17 employing the ratio of nonsynonymous versus synonymous substitution rates has dramatically underestimated the influence of natural selection on molecular evolution. We also find novel evidence from viral sequences to support the co-existence of the crucial role of natural selection in molecular evolution and the ubiquity of neutral mutations. The co-existence has perplexed biologists for decades2,5,7. We then elucidate for the first time the causality between natural selection and the ubiquity of neutral mutations with a novel interpretation of natural selection. This novel interpretation incorporates biochemistry, genetics, epigenetics, physiology, and dynamics. It holds that natural selection acts directly on the overall phenotypic performance of organisms and indirectly on each genomic site or phenotypic trait. It highlights not only restrictions and competitions but also freedom and diversity, besides the overall harmonious development of organisms and human societies. Therefore, this novel interpretation could have far-reaching implications in the natural and social sciences.

Endosomal Trafficking of Two Pore K+ Efflux Channel TWIK2 to Plasmalemma Mediates NLRP3 Inflammasome Activation and Inflammatory Injury

Di, A. — 2022-10-12

Potassium efflux via the two pore K+ channel TWIK2 is a requisite step for the activation of the NLRP3 inflammasome, however it is unclear how the efflux is activated in response to cues. Here we report that during homeostasis, TWIK2 resides in endosomal compartments. TWIK2 is transported by endosomal fusion to the plasmalemma in response to increased extracellular ATP resulting in extrusion of K+ ATP-induced endosomal TWIK2 plasmalemma translocation is regulated by Rab11a. Deleting Rab11a or ATP ligated purinergic receptor P2X7 prevented endosomal fusion with the plasmalemma and K+ efflux and NLRP3 inflammasome activation in macrophages. Adoptive transfer of Rab11a-deleted macrophages into mouse lungs prevented NLRP3 inflammasome activation and inflammatory lung injury. Rab11a-mediated endosomal trafficking in macrophages thus regulates TWIK2 abundance and activity on the cell surface and downstream activation of the NLRP3 inflammasome. Endosomal trafficking of TWIK2 to the plasmalemma is therefore a potential therapy target in acute or chronic inflammatory states.

The mechanism of nicotinamide phosphoribosyltransferase whereby positive allosteric modulation elevates cellular NAD+

Ratia, K. M. — 2022-10-22

In aging and disease, cellular NAD+ is depleted by catabolism to nicotinamide (NAM) and NAD+ supple-mentation is being pursued to enhance human healthspan and lifespan. Activation of nicoti namide phosphoribosyl -transferase (NAMPT), the rate-limiting step in NAD+ biosynthesis, has potential to increase salvage of NAM. Novel NAMPT positive allosteric modulators (N-PAMs) were discovered in addition to demonstration of NAMPT activati on by biogenic phenols. The mechanism of activation was revealed through synthesis of novel chemical probes, new NAMPT co-crystal structures, and enzyme kinetics. Binding to a rear channel in NAMPT regulates NAM binding and turnover, with biochemical observations being replicated by NAD+ measurements in human cells. The mechanism of action of N-PAMs identifies, for the first time, the role of the rear channel in regulation of NAMPT turnover coupled to feedback inhibition by NAM. N-PAM inhibition of low affinity, non-productive NAM binding via the rear channel, causes a right-shif t in KI(NAM) that accompanies an increase in enzyme activity. Conversion of an N-PAM to a high-affinity l igand blocks both high and low affinity NAM binding, ablating enzyme activity. In the presence of an N-PAM, NAMPT boosts NAD+ biosynthesis at higher NAM concentrations, in addition to relieving inhibition by NAD+. Since cellular stress often leads to enhanced catabolism of NAD+ to NAM, this mechanism is relevant to supporting cellular N AD+ levels in aging and disease. The tight regulation of cellular NAMPT is differentially regulated by N-PAMs and other activators, indicating that different classes of pharmacological activators may be engineered for cell and tissue selectivity.

Partial Mimicry of the Microtubule Binding of Tau by Its Membrane Binding

MacAinsh, M. — 2022-11-06

Tau, as typical of intrinsically disordered proteins (IDPs), binds to multiple targets including microtubules and acidic membranes. The latter two surfaces are both highly negatively charged, raising the prospect of mimicry in their binding by tau. The tau-microtubule complex was recently determined by cryo-EM. Here we used molecular dynamics simulations to characterize the dynamic binding of tau K19 to an acidic membrane. This IDP can be divided into three repeats, each containing an amphipathic helix. The three amphipathic helices, along with flanking residues, tether the protein to the membrane interface. The separation between and membrane positioning of the amphipathic helices in the simulations are validated by published EPR data. The membrane contact probabilities of individual residues in tau show both similarities to and distinctions from native contacts with microtubules. In particular, a Lys that is conserved among the repeats forms similar interactions with membranes and with microtubules, as does a conserved Val. This partial mimicry facilitates both the membrane anchoring of microtubules by tau and the transfer of tau from membranes to microtubules.

Parallel Activation of Src and Hif1α Increases Localized Glycolytic ATP Generation for Re-assembly of Endothelial Adherens Junctions

Wang, L. — 2022-11-12

Endothelial adherens junctions (AJs) are critical for the regulation of vascular barrier integrity and undergo dis-assembly during inflammatory injury, thus causing vascular leakiness. AJ re-assembly is thus necessary for restoration of the endothelial barrier following the initial injury. Here we examine the metabolic underpinnings that drive restoration of vascular integrity. In response to inflammatory stimuli, the glycolysis regulatory enzyme PFKFB3 is activated, resulting in a rapid and sustained increase of intracellular glycolytic ATP, especially in the proximity of AJs at the plasma membrane. We engineered a novel chemo-genetic construct (RapT) which allowed for precise temporal control of PFKFB3 recruitment to the plasma membrane. Activation of RapT by rapamycin during the barrier restoration phase increased regional ATP and accelerated AJ re-assembly. Mechanistically, we observed that PFKFB3 is activated through two modes. Src-mediated post-translational phosphorylation rapidly increases PFKFB3 activity. Using another chemo-genetic approach to temporally control Src activity, we found that Src activates PFKFB3 by binding to and phosphorylating it at residues Y175, Y334, and Y363. Tyrosine-phospho-deficient mutants of PFKFB3 at these residues block the glycolytic activation upon inflammatory stimuli. In parallel, elevated reactive oxygen species generated during inflammatory stimulation create pockets of regional hypoxia and allow for increased Hif1-mediated transcription of PFKFB3, leading to sustained glycolytic activation. Moreover, inhibition of PFKFB3 delays AJ reassembly and restoration of vascular integrity both in vitro and in vivo. In conclusion, we show that while inflammatory activation acutely compromises the endothelial barrier, inflammatory signaling also concomitantly generates a metabolic milieu in anticipation of the subsequent re-assembly of AJs and restoration of the vascular barrier.

Toxin-mediated downregulation of absorptive ion transporters NHE3, DRA, and SGLT1 in the colon contributes to diarrhea associated with Clostridioides difficile infection.

Peritore-Galve, F. C. — 2022-11-13

Background & AimClostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis. Two protein toxins, TcdA and TcdB, produced by C. difficile are the major determinants of disease. However, the physiological cause of diarrhea associated with CDI is not well understood. We investigated the effects of CDI on paracellular permeability and apical ion transporters. MethodsWe studied intestinal permeability and apical membrane transporters in female C57BL/6J mice. Ussing chambers were used to measure regional differences in paracellular permeability and ion transporter function in intestinal mucosa. Intestinal tissues were collected from mice and analyzed by immunofluorescence microscopy and RNA-sequencing. ResultsCDI increased intestinal permeability through the size-selective leak pathway in vivo, but permeability was not increased at the sites of pathological damage. Chloride secretion was reduced in the cecum during infection by decreased CaCC function. Infected mice had decreased SGLT1 (also called SLC5A1) activity in the cecum and colon along with diminished apical abundance and an increase in luminal glucose. SGLT1 and DRA (also called SLC26A3) expression was ablated by either TcdA or TcdB, but NHE3 (also called SLC9A3) was decreased in a TcdB-dependent manner. Finally, expression of these three ion transporters was drastically reduced at the transcriptional level. ConclusionsCDI increases intestinal permeability and decreases apical abundance of NHE3, SGLT1, and DRA. This combination may cause a dysfunction in water and solute absorption in the lower gastrointestinal tract, leading to osmotic diarrhea. These findings may open novel pathways for attenuating CDI-associated diarrhea. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=185 HEIGHT=200 SRC="FIGDIR/small/516162v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@e6e23corg.highwire.dtl.DTLVardef@ed6bborg.highwire.dtl.DTLVardef@33adcaorg.highwire.dtl.DTLVardef@d6e359_HPS_FORMAT_FIGEXP M_FIG C_FIG

Highly branched and complementary distributions oflayer 5 and layer 6 auditory corticofugal axons in mouse.

Issa, L. K. — 2022-11-13

The auditory cortex (AC) exerts a powerful, yet heterogeneous, effect on its subcortical targets. Auditory corticofugal projections emanate from distinct bands in layers 5 (L5) and 6 (L6), which have complementary anatomical and physiological properties. While several studies have suggested that corticofugal projections from L5 branch widely, others have suggested that there are multiple, mostly independent sets of L5 corticofugal projections. Even less is known about L6; no studies have examined whether the various L6 corticofugal projections are independent. Therefore, we examined branching patterns of L5 and L6 auditory corticofugal neurons, using the corticocollicular system as an index projection, using both traditional and novel approaches. We first confirmed that dual retrograde injections into the mouse inferior colliculus and auditory thalamus co-labeled subpopulations of L5 and L6 AC neurons. We then used an intersectional approach to selectively re-label L5 or L6 corticocollicular somata and found that both layers sent extensive branches to striatum, amygdala, superior colliculus, thalamus and nuclei of the lateral lemniscus. L5 corticocollicular axons also sent an unpaired projection to the superior olivary complex. Using a novel approach to separately label L5 and L6 axons in the same mouse, we found that L5/6 terminal distributions partially spatially overlapped and that a subset of giant terminals was only found in L5-derived axons. Overall, the high degree of branching and complementarity in the distributions of L5 vs. L6 axons suggest that corticofugal projections should be considered as two widespread systems of projections, rather than a collection of individual projections.

Endothelial Caveolin-1 and CXCL10 promote transcellular migration of autoreactive T cells across the blood-brain barrier

Trevino, T. N. — 2022-11-16

CXCL10 is an interferon-inducible chemokine that can recruit CXCR3+ leukocytes to the central nervous system, leading to neuroinflammation, demyelination, and neuronal losses. How CXCL10 promotes leukocyte extravasation and diapedesis across the blood-brain barrier - formed by brain endothelial cells - is poorly understood. Here, we report that CXCL10 mediates CD4+ T cell migration through the brain endothelial cell cytoplasm (transcellular), but not cell-cell junctions (paracellular), via the vesicular trafficking protein Caveolin-1. Caveolin-1 promotes CXCL10 aggregation into cytoplasmic stores in brain endothelial cells in vitro to provide the local, high concentration necessary for recruitment of CXCR3+ leukocytes. This process also requires LFA-1 activity. In the absence of Caveolin-1, endothelial CXCL10 is secreted, and the local signaling cues are lost. Consistent with our in vitro data, genetic ablation of Caveolin-1 in endothelial cells reduces the severity of active experimental autoimmune encephalomyelitis (EAE), a murine model for multiple sclerosis, by decreasing the infiltration of CXCR3+ T cells into the CNS. Moreover, loss of Caveolin-1 protects against the adoptive transfer of autoreactive T cells. Our findings establish a novel mechanism by which brain endothelial cells utilize Caveolin-1 dependent CXCL10 intracellular stores to license T cells for transcellular migration across the blood-brain barrier.

Female-Specific Pituitary Hypersensitivity to Gonadotropin-Releasing Hormone in a Mouse Model of Chronic Temporal Lobe Epilepsy

Cutia, C. A. — 2022-11-16

Gonadotropin hormone release from the anterior pituitary is critical to regulating reproductive endocrine function. Clinical evidence has documented that people with epilepsy display altered levels of gonadotropin hormones, both acutely following seizures and chronically. Despite this relationship, pituitary function remains a largely understudied avenue in preclinical epilepsy research. Recently, we showed that females in the intrahippocampal kainic acid (IHKA) mouse model of temporal lobe epilepsy were found to display changes in pituitary expression of gonadotropin hormone and GnRH receptor genes. Circulating gonadotropin hormone levels, however, have yet to be measured in an animal model of epilepsy. Here, we evaluated the circulating levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), GnRH receptor (Gnrhr) gene expression, and sensitivity to exogenous GnRH in IHKA males and females. Although no changes in overall dynamics of pulsatile patterns of LH release were found in IHKA mice of either sex, estrus vs. diestrus changes in basal and mean LH levels were larger in IHKA females with prolonged, disrupted estrous cycles. In addition, IHKA females displayed increased pituitary sensitivity to GnRH and higher Gnrhr expression. The hypersensitivity to GnRH was observed on diestrus, but not estrus. Chronic seizure severity was not found to be correlated with LH parameters, and FSH levels were unchanged in IHKA mice. These results indicate that although there are changes in pituitary gene expression and sensitivity to GnRH in IHKA females, there may also be compensatory mechanisms that aid in maintaining gonadotropin release in the state of chronic epilepsy in this model.

Phase-specific signatures of wound fibroblasts and matrix patterns define cancer-associated fibroblast subtypes

Wietecha, M. S. — 2022-11-20

Healing wounds and cancers present remarkable cellular and molecular parallels, but the specific roles of the healing phases are largely unknown. We developed a bioinformatics pipeline to identify genes and pathways that define distinct phases across the time course of healing. Their comparison to cancer transcriptomes revealed that a resolution-phase wound signature is associated with increased severity in skin cancer and enriches for extracellular matrix-related pathways. Comparisons of transcriptomes of early- and late-phase wound fibroblasts vs skin cancer-associated fibroblasts (CAFs) identified an "early-wound" CAF subtype, which localizes to the inner tumor stroma and expresses collagen-related genes that are controlled by the RUNX2 transcription factor. A "late-wound" CAF subtype localizes to the outer tumor stroma and expresses elastin-related genes. Matrix imaging of primary melanoma tissue microarrays validated these matrix signatures and identified collagen- vs elastin-rich niches within the tumor microenvironment, whose spatial organization predicts survival and recurrence. These results identify wound-regulated genes and matrix patterns with prognostic potential in skin cancer.

The binding sites of E2F transcription factor in Drosophila metabolic genes are functionally distinct

Zappia, M. P. — 2022-11-22

The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila. We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase (Pgk), being mostly affected. The loss of E2F regulation on Pgk gene led to a decrease in glycolytic flux, TCA cycle intermediates levels, ATP content and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in Pgk{Delta}E2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in Pgk{Delta}E2F mutants. Moreover, Pgk{Delta}E2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression and development in the Pgk{Delta}E2F animals underscore the importance of E2F regulation on a single E2F target, Pgk.

Why Does Synergistic Activation of WASP, but Not N-WASP, by Cdc42 and PIP2 Require Cdc42 Prenylation?

Dey, S. — 2022-11-24

Human WASP and N-WASP are homologous proteins that require the binding of multiple regulators, including the acidic lipid PIP2 and the small GTPase Cdc42, to relieve autoinhibition before they can stimulate the initiation of actin polymerization. Autoinhibition involves intramolecular binding of the C-terminal acidic and central motifs to an upstream basic region and GTPase binding domain. Little is known about how a single intrinsically disordered protein, WASP or N-WASP, binds multiple regulators to achieve full activation. Here we used molecular dynamics simulations to characterize the binding of WASP and N-WASP with PIP2 and Cdc42. In the absence of Cdc42, both WASP and N-WASP strongly associate with PIP2-containing membranes, through their basic region and also possibly through a tail portion of the N-terminal WH1 domain. The basic region also participates in Cdc42 binding, especially for WASP; consequently Cdc42 binding significantly compromises the ability of the basic region in WASP, but not N-WASP, to bind PIP2. PIP2 binding to the WASP basic region is restored only when Cdc42 is prenylated at the C-terminus and tethered to the membrane. This distinction in the activation of WASP and N-WASP may contribute to their different functional roles.

The Epithelial Na+ Channel UNC-8 promotes an endocytic mechanism that recycles presynaptic components from old to new boutons in remodeling neurons

Cuentas-Condori, A. — 2022-12-01

SummaryPresynaptic terminals are actively relocated during development to refine circuit function, but the underlying cell biological mechanisms are largely unknown. In C. elegans, the presynaptic boutons of GABAergic DD neurons are moved to new locations during early larval development. We show that developmentally regulated expression of a presynaptic Epithelial Na+ Channel (ENaC), UNC-8, promotes a Ca2+-dependent mechanism, resembling Activity-Dependent Bulk Endocytosis (ADBE), that dismantles presynaptic material for reassembly at nascent DD synapses. ADBE normally functions in highly active neurons to accelerate local recycling of synaptic vesicles. We show that DD presynaptic remodeling depends on canonical features of ADBE including elevated intracellular Ca2+, the phosphatase Calcineurin and its targets, dynamin and the F-BAR protein syndapin, and Arp2/3-driven actin polymerization. Thus, our findings suggest that a native mechanism (ADBE) for maintaining neurotransmitter release at local synapses has been repurposed, in this case, to dismantle presynaptic terminals for reassembly at new locations. HighlightsO_LIDeveloping GABAergic neurons dismantle presynaptic terminals for reassembly at new locations. C_LIO_LIThe DEG/ENaC protein, UNC-8, promotes presynaptic disassembly and recycling C_LIO_LICa2+-dependent endocytosis drives presynaptic disassembly and recycling to new boutons C_LI

Structural Model for Self-Limiting β-strand Arrangement Within an Alzheimer's Amyloid-β Oligomer

Gao, Y. — 2022-12-06

Previous reports revealed that sodium dodecyl sulfate near its critical micelle concentration can drive the assembly of A{beta}42 along an oligomeric pathway. This pathway produces a 150 kDa peptide oligomer (approximately 32 peptide molecules or protomers) that does not aggregate further into amyloid fibrils. Solid-state nuclear magnetic resonance (NMR) spectroscopy revealed structural features distinguishing the 150 kDa oligomer from fibrils. A puzzling feature was the coexistence of parallel and antiparallel {beta}-sheets within the oligomer structure. Here we present new atomic-level structural constraints obtained via solid-state NMR spectroscopy, benefitting from improved resolution via sample concentration by ultracentrifugation. In addition, two-dimensional cryo-electron microscopy (cryo-EM) reconstruction revealed a 4-fold symmetric shape. We propose a structural model to rationalize the solid-sate NMR- and cryo-EM-derived structural constraints. This model has a hollow square cylinder shape, with antiparallel {beta}-sheets formed by residues 33-39 lining the inner walls and parallel {beta}-sheets formed by residues 11-22 lining the outer walls. Within successive layers, the outer {beta}-strands on each side of the square cylinder alternate between two forms: one within a U-shaped protomer and another within L-shaped protomer. Molecular dynamics simulations show that, when the oligomer model is embedded in a lipid membrane, ions permeate through the central pore, with cation selectivity. The model further motivates an assembly pathway-based interpretation that may explain why the 150 kDa oligomer does not undergo further aggregation into amyloid fibrils. Significance StatementA{beta} oligomers are thought to be the most toxic species in Alzheimers disease. Their sizes range from 2 to [~]50 protomers. Most published experimental data on A{beta} oligomers indicate that they, like fibrils, are composed of {beta}-sheets, but it is a mystery why any {beta}-sheet aggregate would exist as a stable oligomer without undergoing further aggregation into fibrils. Here, structural constraints from solid-state NMR and cryo-EM led us to an oligomer model with a hollow square cylinder shape capable of conducting ions when embedded in a lipid membrane. Based on the model, we argue that geometric frustration may distinguish the assembly pathway that produces this oligomer from fibril-forming assembly pathways.

Inhibition of translation termination by Drosocin, an antimicrobial peptide from fruit flies

Mangano, K. — 2022-12-11

A 19-amino acid long proline-rich antimicrobial peptide (PrAMP) Drosocin (Dro) is encoded in the fruit fly genome. Native Dro is glycosylated at a specific threonine residue, but the non-glycosylated peptide retains antibacterial activity. Dro shows sequence similarity to several other PrAMPs that bind in the ribosomal nascent peptide exit tunnel and inhibit protein synthesis by varying mechanisms. However, the target and mechanism of action of Dro remain unknown. Here we show that the primary mode of Dro action is inhibition of termination of protein synthesis. Our in vitro and in vivo experiments demonstrate that Dro stalls ribosomes at stop codons, likely sequestering class 1 release factors associated with the terminating ribosome. As the result, Dro strongly promotes readthrough of stop codons at subinhibitory concentrations. The elucidated mode of Dro action allows assigning it as the second member of the type II PrAMPs, of which only one representative, the antimicrobial peptide apidaecin (Api) produced by honeybees, was previously known. However, despite its functional similarity with Api, Dro interacts with the target in a markedly distinct way. The analysis of a comprehensive single-amino acid substitution library of endogenously expressed Dro variants shows that binding to the ribosome involves interactions of multiple amino acid residues distributed through the entire length of the PrAMP. Our data further show that the ribosome-targeting activity of non-glycosylated Dro can be significantly enhanced by single amino acid substitutions illuminating directions for improving its antibacterial properties.

Multimodal Imaging-Based Classification of PTSD Using Data-Driven Computational Approaches: A Multisite Big Data Study from the ENIGMA-PGC PTSD Consortium

Zhu, X. — 2022-12-13

BackgroundCurrent clinical assessments of Posttraumatic stress disorder (PTSD) rely solely on subjective symptoms and experiences reported by the patient, rather than objective biomarkers of the illness. Recent advances in data-driven computational approaches have been helpful in devising tools to objectively diagnose psychiatric disorders. Here we aimed to classify individuals with PTSD versus controls using heterogeneous brain datasets from the ENIGMA-PGC PTSD Working group. MethodsWe analyzed brain MRI data from 3,527 structural-MRI; 2,502 resting state-fMRI; and 1,953 diffusion-MRI. First, we identified the brain features that best distinguish individuals with PTSD from controls (TEHC and HC) using traditional machine learning methods. Second, we assessed the utility of the denoising variational autoencoder (DVAE) and evaluated its classification performance. Third, we assessed the generalizability and reproducibility of both models using leave-one-site-out cross-validation procedure for each modality. ResultsWe found lower performance in classifying PTSD vs. controls with data from over 20 sites (60% test AUC for s-MRI, 59% for rs-fMRI and 56% for d-MRI), as compared to other studies run on single-site data. The performance increased when classifying PTSD from HC without trauma history across all three modalities (75% AUC). The classification performance remained intact when applying the DVAE framework, which reduced the number of features. Finally, we found that the DVAE framework achieved better generalization to unseen datasets compared with the traditional machine learning frameworks, albeit performance was slightly above chance. ConclusionOur findings highlight the promise offered by machine learning methods for the diagnosis of patients with PTSD. The utility of brain biomarkers across three MRI modalities and the contribution of DVAE models for improving generalizability offers new insights into neural mechanisms involved in PTSD. Significance Classifying PTSD from trauma-unexposed healthy controls (HC) using three imaging modalities performed well ([~]75% AUC), but performance suffered markedly when classifying PTSD from trauma-exposed healthy controls (TEHC) using three imaging modalities ([~]60% AUC). Using deep learning for feature reduction (denoising variational auto-encoder; DVAE) dramatically reduced the number of features with no concomitant performance degradation. Utilizing denoising variational autoencoder (DVAE) models improves generalizability across heterogeneous multi-site data compared with the traditional machine learning frameworks

NAD+ biosynthesis as a collateral lethality target for precision oncology

Muller, F. L. — 2022-12-15

Genomic deletion of tumor suppressor genes (TSG) often encompasses neighboring genes which may be members of multi-gene families encoding cell essential functions. These genomic events create targetable cancer-specific vulnerabilities, termed "collateral lethality" as illustrated by homozygous deletion of the glycolytic gene ENO1, which sensitizes glioblastoma (GBM) cells to inhibition of its paralog ENO2. Here, we sought to generalize the concept by validating a second multi-gene family in an unrelated metabolic pathway. Nicotinamide-nucleotide adenylyltransferase (NMNAT) is an essential and (unlike the more extensively studied NAMPT) non-bypassable step in NAD biosynthesis encoded by three paralogs, one of which, NMNAT1, is homozygously deleted as part of the 1p36 tumor suppressor locus in TCGA data of GBM, Cholangiocarcinoma and Hepatocellular carcinoma (with near zero expression of found in several other malignancies). In a glioma cell line (Gli56) with homozygous deletion of NMNAT1 expressing NMNAT2 and NMNAT3, shRNA-mediated knockdown of NMNAT2 is selectively toxic to Gli56 NMNAT1-deleted but not ectopically rescued cells. As NMNAT1 and NMNAT2 are predominantly localized in the nucleus and cytosol, respectively, these data suggest a functionally common pool of cytosolic and nuclear NAD+. Inducible shRNA-mediated extinction of NMNAT2 decreases NAD+ levels and selectively kills NMNAT1-deleted, but not NMNAT1-rescued cells in vitro and eradicates intracranial tumors in vivo. Thus, collateral lethality is a generalizable framework for the development of new classes of targeted agents with an informed clinical development path in cancer. Statement of significanceAn ongoing challenge in precision oncology is the translation of genomic data into actionable therapeutic opportunities with clear clinical benefit. We have demonstrated that genes homozygously deleted by virtue of chromosomal proximity to major tumor suppressor genes can confer cancer-specific vulnerabilities, termed "collateral lethality". While our previous work validated one such collateral lethality target in glycolysis, we now provide empirical in vitro and in vivo evidence that this concept applies to another deleted gene governing an altogether distinct biochemical pathway. The generalization of collateral lethality may expand the spectrum of molecular targets in cancer with a genomically-informed path for accurate clinical development.

CMA-ES-Rosetta: Blackbox optimization algorithm traverses rugged peptide docking energy landscapes

Claussen, E. R. — 2022-12-20

Energy minimization is necessary for virtually all modeling and design tasks and involves traversing extremely rugged energy landscapes. Although the gradient descent based minimization routines in Rosetta have fast runtimes, due to these rugged landscapes, minimization often converges into high-energy local minima. Alternative numerical optimization techniques, such as evolution strategies, are more robust to rugged landscapes and have been shown to be highly successful on a diverse set of problems. Here we explore the Covariance Matrix Adaptation Evolution Strategy (CMA-ES), a state-of-the-art derivative-free optimization algorithm, as a complementary approach to the default minimizer in Rosetta. We used a benchmark of 26 peptides, from the FlexPepDock Benchmark, to assess the performance of three algorithms in Rosetta, specifically, CMA-ES, Rosettas default minimizer, and a Monte Carlo protocol of small backbone perturbations. We test the algorithms performance on their ability to dock an idealized peptide to a series of hotspots residues (i.e. constraints) along a native peptide. Of the three methods, CMA-ES was able to find the lowest energy conformation for 23 out of 26 benchmark peptides. The application of CMA-ES allows for an alternative optimization method for macromolecular modeling problems with rough energy landscapes.

Type I IFN stimulates lymph node stromal cells from adult and old mice during a West Nile virus infection

Bennett, A. K. — 2023-01-05

Advanced age is a significant risk factor during viral infection due to an age-associated decline in the immune response. Older individuals are especially susceptible to severe neuroinvasive disease after West Nile virus (WNV) infection. Previous studies have characterized age-associated defects in hematopoietic immune cells during WNV infection that culminate in diminished antiviral immunity. Situated amongst immune cells in the draining lymph node (DLN) are structural networks of nonhematopoietic lymph node stromal cells (LNSCs). LNSCs are comprised of numerous, diverse subsets, with critical roles in the coordination of robust immune responses. The contributions of LNSCs to WNV immunity and immune senescence are unclear. Here, we examine LNSC responses to WNV within adult and old DLNs. Acute WNV infection triggered cellular infiltration and LNSC expansion in adult. Comparatively, aged DLNs exhibited diminished leukocyte accumulation, delayed LNSC expansion, and altered fibroblast and endothelial cell subset composition, signified by fewer LECs. We established an ex vivo culture system to probe LNSC function. Adult and old LNSCs both recognized an ongoing viral infection primarily through type I IFN signaling. Gene expression signatures were similar between adult and old LNSCs. Aged LNSCs were found to constitutively upregulate immediate early response genes. Collectively, these data suggest LNSCs uniquely respond to WNV infection. We are the first to report age-associated differences in LNSCs on the population- and gene expression-level during WNV infection. These changes may compromise antiviral immunity, leading to increased WNV disease in older individuals.

Single Molecule Mechanics and Kinetics of Cardiac Myosin Interacting with Regulated Thin Filaments

Clippinger Schulte, S. R. — 2023-01-10

The cardiac cycle is a tightly regulated process wherein the heart generates force to pump blood to the body during systole and then relaxes during diastole. Disruption of this finely tuned cycle can lead to a range of diseases including cardiomyopathies and heart failure. Cardiac contraction is driven by the molecular motor myosin, which pulls regulated thin filaments in a calcium-dependent manner. In some muscle and non-muscle myosins, regulatory proteins on actin tune the kinetics, mechanics, and load dependence of the myosin working stroke; however, it is not well understood whether or how thin filament regulatory proteins tune the mechanics of the cardiac myosin motor. To address this critical gap in knowledge, we used single-molecule techniques to measure the kinetics and mechanics of the substeps of the cardiac myosin working stroke in the presence and absence of thin filament regulatory proteins. We found that regulatory proteins gate the calcium-dependent interactions between myosin and the thin filament. At physiologically relevant ATP concentrations, cardiac myosins mechanics and unloaded kinetics are not affected by thin filament regulatory proteins. We also measured the load-dependent kinetics of cardiac myosin at physiologically relevant ATP concentrations using an isometric optical clamp, and we found that thin filament regulatory proteins do not affect either the identity or magnitude of myosins primary load-dependent transition. Interestingly, at low ATP concentrations, thin filament regulatory proteins have a small effect on actomyosin dissociation kinetics, suggesting a mechanism beyond simple steric blocking. These results have important implications for both disease modeling and computational models of muscle contraction. Significance StatementHuman heart contraction is powered by the molecular motor {beta}-cardiac myosin, which pulls on thin filaments consisting of actin and the regulatory proteins troponin and tropomyosin. In some muscle and non-muscle systems, these regulatory proteins tune the kinetics, mechanics, and load dependence of the myosin working stroke. Despite having a central role in health and disease, it is not well understood whether the mechanics or kinetics of {beta}-cardiac myosin are affected by regulatory proteins. We show that regulatory proteins do not affect the mechanics or load-dependent kinetics of the working stroke at physiologically relevant ATP concentrations; however, they can affect the kinetics at low ATP concentrations, suggesting a mechanism beyond simple steric blocking. This has important implications for modeling of cardiac physiology and diseases.

MESH-FREE HIGH-RESOLUTION SIMULATION OF CEREBROCORTICAL OXYGENSUPPLY WITH FAST FOURIER PRECONDITIONING

Linninger, A. — 2023-01-10

Oxygen transfer from blood vessels to cortical brain tissue is representative of a class of problems with mixed-domain character. Large-scale efficient computation of tissue oxygen concentration is dependent on the manner in which the tubular network of blood vessels is coupled to the tissue mesh. Models which explicitly resolve the interface between the tissue and vasculature with a contiguous mesh are prohibitively expensive for very dense cerebral microvasculature. We propose a mixed-domain mesh-free technique whereby a vascular anatomical network (VAN) represented as a thin directed graph serves for convection of blood oxygen, and the surrounding extravascular tissue is represented as a Cartesian grid of 3D voxels throughout which oxygen is transported by diffusion. We split the network and tissue meshes by the Schur complement method of domain decomposition to obtain a reduced set of system equations for the tissue oxygen concentration. The use of a Cartesian grid allows the corresponding matrix equation to be solved approximately with a fast Fourier transform based Poisson solver, which serves as an effective preconditioner for Krylov subspace iteration. The performance of this method enables the steady state simulation of cortical oxygen perfusion for anatomically accurate vascular networks down to single micron resolution without the need for supercomputers. Practitioner PointsO_LIWe present a novel mixed-domain framework for efficiently modeling O2 extraction kinetics in the brain. C_LIO_LIModel equations are generated by graph-theoretic methods for mixed domains. C_LIO_LIDual mesh domain decomposition with FFT preconditioning yields very fast simulation times for extremely high spatial resolution. C_LI

Spin-coupled electron densities of iron-sulfur cluster imaged by in situ serial Laue diffraction

Ren, Z. — 2023-01-10

Iron-sulfur clusters are inorganic cofactors found in many proteins involved in fundamental biological processes including DNA processing. The prokaryotic DNA repair enzyme PhrB, a member of the protein family of cryptochromes and photolyases, carries a four-iron-four-sulfur cluster [4Fe4S] in addition to the catalytic cofactor flavin adenine dinucleotide (FAD) and a second pigment 6,7-dimethyl-8-ribityllumazine (DMRL). The light-induced redox reactions of this multi-cofactor protein complex were recently shown as two interdependent photoreductions of FAD and DMRL mediated by the [4Fe4S] cluster functioning as an electron cache to hold a fine balance of electrons. Here, we apply the more traditional temperature-scan cryo-trapping technique in protein crystallography and the newly developed technology of in situ serial Laue diffraction at room temperature. These diffraction methods in dynamic crystallography enable us to capture strong signals of electron density changes in the [4Fe4S] cluster that depict quantized electronic movements. The mixed valence layers of the [4Fe4S] cluster due to spin coupling and their dynamic responses to light illumination are observed directly in our difference maps between its redox states. These direct observations of the quantum effects in a protein bound iron-sulfur cluster have thus opened a window into the mechanistic understanding of metal clusters in biological systems.

Phylogenetic modeling of enhancer shifts in African mole-rats reveals regulatory changes associated with tissue-specific traits

Parey, E. — 2023-01-10

Changes in gene regulation have long been thought to underlie most phenotypic differences between species. Subterranean rodents, and in particular the naked mole-rat, have attracted substantial attention due to their proposed phenotypic adaptations, which include hypoxia tolerance, metabolic changes and cancer resistance. However, it is largely unknown what regulatory changes may associate with these phenotypic traits, and whether these are unique to the naked mole-rat, the mole-rat clade or also present in other mammals. Here, we investigate regulatory evolution in heart and liver from two African mole-rat species and two rodent outgroups using genome-wide epigenomic profiling. First, we adapted and applied a phylogenetic modeling approach to quantitatively compare epigenomic signals at orthologous regulatory elements, and identified thousands of promoter and enhancer regions with differential epigenomic activity in mole-rats. These elements associate with known mole-rat adaptation in metabolic and functional pathways, and suggest candidate genetic loci that may underlie mole-rat innovations. Second, we evaluated ancestral and species-specific regulatory changes in the study phylogeny, and report several candidate pathways experiencing stepwise remodeling during the evolution of mole-rats - such as the insulin and hypoxia response pathways. Third, we report non-orthologous regulatory elements overlap with lineage-specific repetitive elements and appear to modify metabolic pathways by rewiring of HNF4 and RAR/RXR transcription factor binding sites in mole-rats. These comparative analyses reveal how mole-rat regulatory evolution informs previously reported phenotypic adaptations. Moreover, the phylogenetic modeling framework we propose here improves upon the state-of-the-art by addressing known limitations of inter-species comparisons of epigenomic profiles, and has broad implications in the field of comparative functional genomics.

Physical mechanisms of red blood cell splenic filtration

Moreau, A. — 2023-01-10

The splenic interendothelial slits fulfill the essential function of continuously filtering red blood cells (RBCs) from the bloodstream to eliminate abnormal and aged cells. To date, the process by which 8 {micro}m RBCs pass through 0.3 {micro}m-wide slits remains enigmatic. Does the slit caliber increase during RBC passage as sometimes suggested? Here, we elucidated the mechanisms that govern the RBC retention or passage dynamics in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with sub-micron wide physiologically calibrated slits. We observed that healthy RBCs pass through 0.28 {micro}m-wide rigid slits at 37{degrees}C. To achieve this feat, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, the cells with a low surface area-to-volume ratio (28 % of RBCs in a 0.4 {micro}m-wide slit) must locally unfold their spectrin cytoskeleton inside the slit. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through the slits follows a -1 and -3 power law with in-slit pressure drop and slip width, respectively. This law is similar to that of a Newtonian fluid in a 2D Poiseuille flow, showing that the dynamics of RBCs is controlled by their cytoplasmic viscosity. Altogether, our results show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in-vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery. Significance StatementSplenic filtration of red blood cells through narrow interendothelial slits remains poorly understood despite its physiological significance as experiments and imaging of red cells passing through the slits are lacking. Here, we coupled live imaging, biomimetic submicron-fluidics, and multiscale modeling to quantify passage conditions. Remarkably, healthy 8-{micro}m cells can pass through 0.28-{micro}m slits at body temperature. This event is conditioned to cells being able to deform into two tether-connected equal spheres and, in limiting cases, to unfold their spectrin cytoskeleton. We showed that cells behave like a Newtonian fluid and that their dynamics is controlled by the inner fluid viscosity. We thus propose an in-vitro and in-silico approach to quantify splenic clearance of diseased cells and cells engineered for transfusion and drug delivery.

Periosteal skeletal stem cells can migrate into the bone marrow and support hematopoiesis after injury

Marchand, T. — 2023-01-13

Skeletal stem cells have been isolated from various tissues, including periosteum and bone marrow, where they exhibit key functions in bone biology and hematopoiesis, respectively. The role of periosteal skeletal stem cells in bone regeneration and healing has been extensively studied, but their ability to contribute to the bone marrow stroma is still under debate. In the present study, we characterized a whole bone transplantation model that mimics the initial bone marrow necrosis and fatty infiltration seen after injury. Using this model and a lineage tracing approach, we observed the migration of periosteal skeletal stem cells into the bone marrow after transplantation. Once in the bone marrow, periosteal skeletal stem cells are phenotypically and functionally reprogrammed into bone marrow mesenchymal stem cells that express high levels of hematopoietic stem cell niche factors such as Cxcl12 and Kitl. In addition, using ex vivo and in vivo approaches, we found that periosteal skeletal stem cells are more resistant to acute stress than bone marrow mesenchymal stem cells. These results highlight the plasticity of periosteal skeletal stem cells and their potential role in bone marrow regeneration after bone marrow injury.

An Open-Source Platform for Head-Fixed Operant and Consummatory Behavior

Gordon-Fennell, A. — 2023-01-16

Head-fixed behavioral experiments in rodents permit unparalleled experimental control, precise measurement of behavior, and concurrent modulation and measurement of neural activity. Here we present OHRBETS (Open-Source Head-fixed Rodent Behavioral Experimental Training System; pronounced Orbitz), a low-cost, open-source ecosystem of hardware and software to flexibly pursue the neural basis of a variety of motivated behaviors. Head-fixed mice tested with OHRBETS displayed operant conditioning for caloric reward that replicates core behavioral phenotypes observed during freely moving conditions. OHRBETS also permits for optogenetic intracranial self-stimulation under positive or negative operant conditioning procedures and real-time place preference behavior, like that observed in freely moving assays. In a multi-spout brief-access consumption task, mice displayed licking as a function of concentration of sucrose, quinine, and sodium chloride, with licking modulated by homeostatic or circadian influences. Finally, to highlight the functionality of OHRBETS, we measured mesolimbic dopamine signals during the multi-spout brief-access task that display strong correlations with relative solution value and magnitude of consumption. All designs, programs, and instructions are provided freely online. This customizable ecosystem enables replicable operant and consummatory behaviors and can be incorporated with methods to perturb and record neural dynamics in vivo. Impact StatementA customizable open-source hardware and software ecosystem for conducting diverse head-fixed behavioral experiments in mice.

IFI207, a young and fast-evolving antiviral factor, stabilizes STING

Moran, E. A. — 2023-01-19

Mammalian ALR proteins bind nucleic acids and initiate production of type I interferons or inflammasome assembly, thereby contributing to host innate immunity. ALRs are encoded at a single genetic locus. In mice, the Alr locus is highly polymorphic at the sequence and copy number level. We suggest that one rapidly evolving member of the Alr family, Ifi207, was introduced to the Mus genome by a recent recombination event. Ifi207 has a large, distinctive repeat region that differs in sequence and length in different Mus strains. We show that IFI207 plays a key role in the STING-mediated response to cGAMP, DNA, and MLV, and that IFI207 controls MLV infection in vivo. Uniquely, IFI207 acts by stabilizing STING protein via its repeat region. Our studies suggest that under the pressure of host-pathogen coevolution, in a dynamic locus such as the Alr, recombination between gene family members creates new genes with novel and essential functions that play diverse roles in biological processes.

Sex and estrous cycle stage shape left-right asymmetry in chronic hippocampal seizures in mice

Cutia, C. A. — 2023-01-21

Lateralization of hippocampal function is indicated by varied outcomes of patients with neurological disorders that selectively affect one hemisphere of this structure, such as temporal lobe epilepsy (TLE). The intrahippocampal kainic acid (IHKA) injection model of TLE allows for targeted damage to the left or right hippocampus, enabling systematic comparison of effects of left-right asymmetry on seizure and non-seizure outcomes. Although varying non-seizure phenotypic outcomes based on injection side in dorsal hippocampus were recently evaluated in this model, differences in chronic seizure patterns in left- (IHKA-L) vs. right-injected (IHKA-R) IHKA animals have yet to be evaluated. Here, we evaluated hippocampal seizure incidence in male and female IHKA-L and IHKA-R mice. Females displayed increased electrographic seizure activity compared to males at both 2 months and 4 months post-injection (mpi). In addition, IHKA-L females showed higher seizure frequency than IHKA-R on diestrus and estrus at 2 mpi, but seizure duration and time in seizures were only higher in IHKA-L females on diestrus. These cycle stage-associated changes, however, did not persist to 4 mpi. Furthermore, this lateralized difference in seizure burden was not observed in males. These results indicate for the first time that the side of IHKA injection can shape chronic electrographic seizure burden. Overall, these results demonstrate a female-specific left-right asymmetry in hippocampal function can interact with estrous cycle stage to shape chronic seizures in mice with epilepsy, with implications for neural activity and behavior in both normal and disease states.

BRWD1 orchestrates chromatin topology by converting static to dynamic cohesin complexes

Mandal, M. — 2023-01-23

Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative selection and antigen receptor gene recombination1. Transitions between each state require large, coordinated changes in epigenetic landscapes and transcriptional programs2,3. How this occurs remains unclear. Herein, we demonstrate that in small pre-B cells, the lineage and stagespecific epigenetic reader Bromodomain and WD Repeating Containing Protein 1 (BRWD1)2,4 reorders three-dimensional chromatin topology to affect transition between proliferative and gene recombination molecular programs. BRWD1 regulated the switch between poised and active enhancers interacting with promoters and coordinated this with Igk locus contraction. BRWD1 did so by converting chromatin-bound static cohesin to dynamic complexes competent to mediate long-range looping. Remarkably, ATP depletion recapitulated cohesin distributions observed in Brwd1-/- cells. Therefore, in small pre-B cells, cohesin conversion is the main energetic mechanism dictating where dynamic looping occurs in the genome. Our findings provide a new mechanism of cohesin regulation and reveal how cohesin function can be dictated by lineage contextual mechanisms to facilitate specific cell fate transitions.

Co-transplantation with mesenchymal stem cells and endothelial cells improvise islet engraftment and survival in STZ treated hyperglycemic mice

Naqvi, R. A. — 2023-01-25

Though intra-portal islet transplantation demonstrated as best suited strategy for the reversal of hyperglycemia without the threat of iatrogenic hyperglycemia in type 1 diabetes (T1D) in patients, the inferior quality of post-transplantation (tx) vascularization needs to be addressed for the maximization of post-tx islet survival. Therefore, in this study, we have first generated MSCs and endothelial progenitor cells (EPC) from mice bone marrow by in house optimized protocol and then 3-D co-cultured them with mice islets. Secretion of in the culture supernatant suggested the pro-angiogenic nature of 3D cultured mice islets. After 5 days post-tx of these pro-angiogenic islets in the omental pouch of syngeneic mice led to: 1) restoration of normoglycemia, 2) secretion of mouse C-peptide and 3) induction of angiogenic factors after 3 days of post-tx. The induction of angiogenic factors was done by RT-qPCR of omental biopsies. Importantly, pro-angiogenic islet recipient mice also demonstrated the clearance of glucose within 75 min, reflecting their efficient function and engraftment. Our results highlights needs of 3-D co-culture islets for superior quality post-tx islet vasculature and better engraftment - crux to improvise the challenges associated with post-tx islet vascularization and functions.

Sexually Dimorphic Role for Insular Perineuronal Nets in Aversion-Resistant Ethanol Consumption

Carvalho, L. M. — 2023-01-28

Compulsive alcohol drinking is a key symptom of alcohol use disorder (AUD) that is particularly resistant to treatment. An understanding of the biological factors that underly compulsive drinking will allow for the development of new therapeutic targets for AUD. One animal model of compulsive alcohol drinking involves the addition of bitter-tasting quinine to an ethanol solution and measuring the willingness of the animal to consume ethanol despite the aversive taste. Previous studies have demonstrated that this type of aversion-resistant drinking is modulated in the insular cortex of male mice by specialized condensed extracellular matrix known as perineuronal nets (PNNs), which form a lattice-like structure around parvalbumin-expressing neurons in the cortex. Several laboratories have shown that female mice exhibit higher levels of aversion-resistant ethanol intake but the role of PNNs in females in this behavior has not been examined. Here we compared PNNs in the insula of male and female mice and determined if disrupting PNNs in female mice would alter aversion-resistant ethanol intake. PNNs were visualized in the insula by fluorescent labeling with Wisteria floribunda agglutinin (WFA) and disrupted in the insula by microinjecting chondroitinase ABC, an enzyme that digests the chondroitin sulfate glycosaminoglycan component of PNNs. Mice were tested for aversion-resistant ethanol consumption by the addition of sequentially increasing concentrations of quinine to the ethanol in a two-bottle choice drinking in the dark procedure. PNN staining intensity was higher in the insula of female compared to male mice, suggesting that PNNs in females might contribute to elevated aversion-resistant drinking. However, disruption of PNNs had limited effect on aversion-resistant drinking in females. In addition, activation of the insula during aversion-resistant drinking, as measured by c-fos immunohistochemistry, was lower in female mice than in males. Taken together, these results suggest that neural mechanisms underlying aversion-resistant ethanol consumption differ in males and females.

Organism-Wide Analysis of Sepsis Reveals Mechanisms of Systemic Inflammation

Takahama, M. — 2023-02-02

Sepsis is a systemic response to infection with life-threatening consequences. Our understanding of the impact of sepsis across organs of the body is rudimentary. Here, using mouse models of sepsis, we generate a dynamic, organism-wide map of the pathogenesis of the disease, revealing the spatiotemporal patterns of the effects of sepsis across tissues. These data revealed two interorgan mechanisms key in sepsis. First, we discover a simplifying principle in the systemic behavior of the cytokine network during sepsis, whereby a hierarchical cytokine circuit arising from the pairwise effects of TNF plus IL-18, IFN-{gamma}, or IL-1{beta} explains half of all the cellular effects of sepsis on 195 cell types across 9 organs. Second, we find that the secreted phospholipase PLA2G5 mediates hemolysis in blood, contributing to organ failure during sepsis. These results provide fundamental insights to help build a unifying mechanistic framework for the pathophysiological effects of sepsis on the body.

Genetic control of mRNA splicing as a potential mechanism for incomplete penetrance of rare coding variants

Einson, J. — 2023-01-31

Exonic variants present some of the strongest links between genotype and phenotype. However, these variants can have significant inter-individual pathogenicity differences, known as variable penetrance. In this study, we propose a model where genetically controlled mRNA splicing modulates the pathogenicity of exonic variants. By first cataloging exonic inclusion from RNA-seq data in GTEx v8, we find that pathogenic alleles are depleted on highly included exons. Using a large-scale phased WGS data from the TOPMed consortium, we observe that this effect may be driven by common splice-regulatory genetic variants, and that natural selection acts on haplotype configurations that reduce the transcript inclusion of putatively pathogenic variants, especially when limiting to haploinsufficient genes. Finally, we test if this effect may be relevant for autism risk using families from the Simons Simplex Collection, but find that splicing of pathogenic alleles has a penetrance reducing effect here as well. Overall, our results indicate that common splice-regulatory variants may play a role in reducing the damaging effects of rare exonic variants.

Predicting the Sequence-Dependent Backbone Dynamics of Intrinsically Disordered Proteins

Qin, S. — 2023-02-03

How the sequences of intrinsically disordered proteins (IDPs) code for functions is still an enigma. Dynamics, in particular residue-specific dynamics, holds crucial clues. Enormous efforts have been spent to characterize residue-specific dynamics of IDPs, mainly through NMR spin relaxation experiments. Here we present a sequence-based method, SeqDYN, for predicting residue-specific backbone dynamics of IDPs. SeqDYN employs a mathematical model with 21 parameters: one is a correlation length and 20 are the contributions of the amino acids to slow dynamics. Training on a set of 45 IDPs reveals aromatic, Arg, and long-branched aliphatic amino acids as the most active in slow dynamics whereas Gly and short polar amino acids as the least active. SeqDYN predictions not only provide an accurate and insightful characterization of sequence-dependent IDP dynamics but may also serve as indicators in a host of biophysical processes, including the propensities of IDP sequences to undergo phase separation.

The seminal odorant binding protein Obp56g is required for mating plug formation and male fertility in Drosophila melanogaster

Brown, N. — 2023-02-07

In Drosophila melanogaster and other insects, the seminal fluid proteins (SFPs) and male sex pheromones that enter the female with sperm during mating are essential for fertility and induce profound post-mating effects on female physiology and behavior. The SFPs in D. melanogaster and other taxa include several members of the large gene family known as odorant binding proteins (Obps). Previous work in Drosophila has shown that some Obp genes are highly expressed in the antennae and can mediate behavioral responses to odorants, potentially by binding and carrying these molecules to odorant receptors. These observations have led to the hypothesis that the seminal Obps might act as molecular carriers for pheromones or other compounds important for male fertility in the ejaculate, though functional evidence in any species is lacking. Here, we used RNAi and CRISPR/Cas9 generated mutants to test the role of the seven seminal Obps in D. melanogaster fertility and the post-mating response (PMR). We found that Obp56g is required for male fertility and the induction of the PMR, whereas the other six genes had no effect on fertility when mutated individually. Obp56g is expressed in the males ejaculatory bulb, an important tissue in the reproductive tract that synthesizes components of the mating plug. We found males lacking Obp56g fail to form a mating plug in the mated females reproductive tract, leading to ejaculate loss and reduced sperm storage. We also examined the evolutionary history of these seminal Obp genes, as several studies have documented rapid evolution and turnover of SFP genes across taxa. We found extensive lability in gene copy number and evidence of positive selection acting on two genes, Obp22a and Obp51a. Comparative RNAseq data from the male reproductive tract of multiple Drosophila species revealed that Obp56g shows high male reproductive tract expression only in species of the melanogaster and obscura groups, though conserved head expression in all species tested. Together, these functional and expression data suggest that Obp56g may have been co-opted for a reproductive function over evolutionary time.

An Unexpected Role of Neutrophils in Clearing Apoptotic Hepatocytes In Vivo

Cao, L. — 2023-02-08

Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.

Modulation of Retinal Inflammation Delays Degeneration in a Mouse Model of Geographic Atrophy

Ridley, R. B. — 2023-02-09

The advanced form of AMD, geographic atrophy, is associated with increased RPE oxidative stress and chronic inflammation. Here we evaluated the effects of delivering an anti-inflammatory viral gene by an AAV-vector in a mouse model of geographic atrophy. We measured changes in retinal function, structure, and morphology over nine months with electroretinography, optical coherence tomography, and fundoscopy, respectively. In addition, we used retinal tissue to quantify changes in markers of inflammation by multiplex ELISA, RT-qPCR, and immunofluorescence staining. Our AAV significantly delayed the loss of retinal function and structure and decreased retinal inflammation compared to the control AAV treatment. Our results suggest that modulating retinal inflammation could significantly slow the progression of geographic atrophy.

Seroprevalence of IgG Antibody against SARS-CoV-2 Nucleocapsid protein and Associated Risk Factors

Chowdhury, Y. F. — 2023-02-09

Estimation of antibody development against SARS-CoV-2 is essential means for understanding the immune response against the virus. We reported IgG antibody development status against Nucleocapsid protein of the virus and compared with lifestyle (health and food habits), co-existing diseases, vaccination and COVID-19 infection status. ELISA (Enzyme Linked Immunosorbent Assay) was performed to assess IgG antibodies targeted against the Nucleocapsid protein of SARS-CoV-2 in participants (n=500). In this seroprevalence study, serological data were estimated for a period of 10 months in the participants who were aged 10 years and above. Sociodemographic and risk factors related data were collected through a written questionnaire and chi-square test was performed to determine the association with seropositivity. The overall seroprevalence of anti-SARS-CoV-2 antibodies among the study subjects was 47.8%. Estimates were highest among the participants of 21-40 years old (55.1%), and lowest in older aged (>60 years) participants (39.5%). Among the Sinopharm vaccinated individuals 81.8% had developed anti-Nucleocapsid antibody. Physical exercise and existence of comorbidities like hypertension and diabetes were the distinguishing factors between seropositive and seronegative individuals. Seropositivity rate largely varied among symptomatic (67%) and asymptomatic (33.1%) COVID-19 infected participants. The findings suggest that residents of Dhaka city had a higher prevalence of anti-nucleocapsid antibody in the second year of the pandemic. This indicates the improvement of immunological status among the population. Finally, the study emphasizes on maintaining active and healthy lifestyle to improve immunity. However, the absence of IgG antibodies in many cases of COVID-19 infected individuals suggests that antibodies wane with time. Key messagesO_LIThe overall seroprevalence of anti-Nucleocapsid IgG among the study subjects was determined to be 47.8%. C_LIO_LIAge, regular physical exercise, existence of comorbidities were the identified parameters associated with seroprevalence. C_LIO_LIThis study observed lower prevalence of Anti-Nucleocapsid antibody among asymptomatic cases of COVID-19 infected individuals compared to symptomatic cases. C_LI

In vivo evaluation of the effect of sickle cell hemoglobin S, C and therapeutic transfusion on erythrocyte metabolism and cardiorenal dysfunction

D'Alessandro, A. — 2023-02-14

Despite a wealth of exploratory plasma metabolomics studies in sickle cell disease (SCD), no study to date has evaluate a large and well phenotyped cohort to compare the primary erythrocyte metabolome of hemoglobin SS, SC and transfused AA red blood cells (RBCs) in vivo. The current study evaluates the RBC metabolome of 587 subjects with sickle cell sickle cell disease (SCD) from the WALK-PHaSST clinical cohort. The set includes hemoglobin SS, hemoglobin SC SCD patients, with variable levels of HbA related to RBC transfusion events, and HbF related to hydroxyurea therapy. Here we explore the modulating effects of genotype, age, sex, severity of hemolysis, and hydroxyurea and transfusion therapy on sickle RBC metabolism. Data - collated in an online portal - show that the Hb SS genotype is associated with significant alterations of RBC acylcarnitines, pyruvate, sphingosine 1-phosphate, creatinine, kynurenine and urate metabolism. Surprisingly, the RBC metabolism of SC RBCs is dramatically different from SS, with all glycolytic intermediates significantly elevated in SS RBCs, with the exception of pyruvate. This result suggests a metabolic blockade at the ATP-generating phosphoenolpyruvate to pyruvate step of glycolysis, which is catalyzed by redox-sensitive pyruvate kinase. Increasing in vivo concentrations of HbA improved glycolytic flux and normalized the HbS erythrocyte metabolome. An unexpectedly limited metabolic effect of hydroxyurea and HbF was observed, possibly related to the modest induction of HbF in this cohort. The metabolic signature of HbS RBCs correlated with the degree of steady state hemolytic anemia, cardiovascular and renal dysfunction and mortality. Key pointsO_LIIn vivo dysregulation of RBC metabolism by HbS is evaluated by metabolic profiling of 587 patients with variable HbA, HbC and HbF levels; C_LIO_LIRBC acyl-carnitines, urate, pyruvate metabolism, S1P, kynurenine relate to hemolysis and cardiorenal dysfunction, respond to transfusion; C_LI

kakapo: Easy extraction and annotation of genes from raw RNA-seq reads

Ramanauskas, K. — 2023-02-15

kakapo (k[a]k[a]p[o]) is a python-based pipeline that allows users to extract and assemble one or more specified genes or gene families. It flexibly uses original RNA-seq read or GenBank SRA accession inputs without performing assembly of entire transcriptomes. The pipeline identifies open reading frames in the assembled gene transcripts and annotates them. It optionally filters raw reads for ribosomal, plastid, and mitochondrial reads, or reads belonging to non-target organisms (e.g., viral, bacterial, human). kakapo can be employed to extract arbitrary loci, such as those commonly used for phylogenetic inference in systematics or candidate genes and gene families in phylogenomic and metagenomic studies. We provide example applications and discuss how its use can offset the declining value of the GenBanks single-gene databases and help assemble datasets for a variety of phylogenetic analyses.

Adolescent Intermittent Ethanol Exposure Alters Exploratory and Affective Behaviors, and Cerebellar Grin2B Expression in Adulthood

Healey, K. — 2023-02-14

Binge drinking is one of the most common patterns (more than 90%) of alcohol consumption by young people. During adolescence, the brain undergoes maturational changes that influence behavioral control and affective behaviors, such as cerebellar brain volume and function in adulthood. We investigated long-term impacts of adolescent binge ethanol exposure on affective and exploratory behaviors and cerebellar gene expression in adult male and female mice. Further, the cerebellum is increasingly recognized as a brain region integrating a multitude of behaviors that span from the traditional primary sensory-motor to affective functions, such as anxiety and stress reactivity. Therefore, we investigated the persistent effects of adolescent intermittent ethanol (AIE) on exploratory and affective behaviors and began to elucidate the role of the cerebellum in these behaviors through excitatory signaling gene expression. We exposed C57BL/6J mice to AIE or air (control) vapor inhalation from postnatal day 28-42. After prolonged abstinence (>34 days), in young adulthood (PND 77+) we assessed behavior in the open field, light/dark, tail suspension, and forced swim stress tests to determine changes in affective behaviors including anxiety-like, depressive-like, and stress reactivity behavior. Excitatory signaling gene mRNA levels of fragile X messenger ribonucleoprotein (FMR1), glutamate receptors (Grin2a, Grin2B and Grm5) and excitatory synaptic markers (PSD-95 and Eaat1) were measured in the cerebellum of adult control and AIE-exposed mice. AIE-exposed mice showed decreased exploratory behaviors in the open field test (OFT) where both sexes show reduced ambulation, however only females exhibited a reduction in rearing. Additionally, in the OFT, AIE-exposed females also exhibited increased anxiety-like behavior (entries to center zone). In the forced swim stress test, AIE-exposed male mice, but not females, spent less time immobile compared to their same-sex controls, indicative of sex-specific changes in stress reactivity. Male and female AIE-exposed mice showed increased Grin2B (Glutamate Ionotropic Receptor NMDA Type Subunit 2B) mRNA levels in the cerebellum compared to their same-sex controls. Together, these data show that adolescent binge-like ethanol exposure altered both exploratory and affective behaviors in a sex-specific manner and modified cerebellar Grin2B expression in adult mice. This indicates the cerebellum may serve as an important brain region that is susceptible to long-term molecular changes after AIE. HighlightsO_LIAdolescent intermittent ethanol (AIE) exposure decreased exploratory behavior in adult male and female mice. C_LIO_LIIn females, but not males, AIE increased anxiety-like behavior. C_LIO_LIIn males, but not females, AIE reduced stress reactivity in adulthood. C_LIO_LIThese findings indicate sex differences in the enduring effects of AIE on exploratory and affective behaviors. C_LIO_LICerebellar Grin2B mRNA levels were increased in adulthood in both male and female AIE-exposed mice. C_LIO_LIThese findings add to the small, but growing literature on behavioral AIE effects in mice, and establish cerebellar excitatory synaptic gene expression as an enduring effect of adolescent ethanol exposure. C_LI

Enhanced simulations of whole-brain dynamics using hybrid resting-state structural connectomes

Manos, T. — 2023-02-16

The human brain, composed of billions of neurons and synaptic connections, is an intricate network coordinating a sophisticated balance of excitatory and inhibitory activity between brain regions. The dynamical balance between excitation and inhibition is vital for adjusting neural input/output relationships in cortical networks and regulating the dynamic range of their responses to stimuli. To infer this balance using connectomics, we recently introduced a computational framework based on the Ising model, first developed to explain phase transitions in ferromagnets, and proposed a novel hybrid resting-state structural connectome (rsSC). Here, we show that a generative model based on the Kuramoto phase oscillator can be used to simulate static and dynamic functional connectomes (FC) with rsSC as the coupling weight coefficients, such that the simulated FC well aligns with the observed FC when compared to that simulated with traditional structural connectome. Simulations were performed using the open source framework The Virtual Brain on High Performance Computing infrastructure.

Hepatitis delta virus RNA decline post inoculation in human NTCP transgenic mice is biphasic

Maya, S. — 2023-02-17

Background and AimsChronic infection with hepatitis B and hepatitis delta viruses (HDV) is considered the most serious form of viral hepatitis due to more severe manifestations of and accelerated progression to liver fibrosis, cirrhosis, and hepatocellular carcinoma. There is no FDA-approved treatment for HDV and current interferon-alpha treatment is suboptimal. We characterized early HDV kinetics post inoculation and incorporated mathematical modeling to provide insights into host-HDV dynamics. MethodsWe analyzed HDV RNA serum viremia in 192 immunocompetent (C57BL/6) and immunodeficient (NRG) mice that did or did not transgenically express the HDV receptor - human sodium taurocholate co-transporting peptide (hNTCP). ResultsKinetic analysis indicates an unanticipated biphasic decline consisting of a sharp first-phase and slower second-phase decline regardless of immunocompetence. HDV decline after re-inoculation again followed a biphasic decline; however, a steeper second-phase HDV decline was observed in NRG-hNTCP mice compared to NRG mice. HDV-entry inhibitor bulevirtide administration and HDV re-inoculation indicated that viral entry and receptor saturation are not major contributors to clearance, respectively. The biphasic kinetics can be mathematically modeled by assuming the existence of a non-specific binding compartment with a constant on/off-rate and the steeper second-phase decline by a loss of bound virus that cannot be returned as free virus to circulation. The model predicts that free HDV is cleared with a half-life of 18 minutes (standard error, SE: 2.4), binds to non-specific cells with a rate of 0.06 hour-1 (SE: 0.03), and returns as free virus with a rate of 0.23 hour-1 (SE: 0.03). ConclusionsUnderstanding early HDV-host kinetics will inform pre-clinical therapeutic kinetic studies on how the efficacy of anti-HDV therapeutics can be affected by early kinetics of viral decline. LAY SUMMARYThe persistence phase of HDV infection has been studied in some animal models, however, the early kinetics of HDV in vivo is incompletely understood. In this study, we characterize an unexpectedly HDV biphasic decline post inoculation in immunocompetent and immunodeficient mouse models and use mathematical modeling to provide insights into HDV-host dynamics. Understanding the kinetics of viral clearance in the blood can aid pre-clinical development and testing models for anti-HDV therapeutics.

The Conserved C-terminal End Segment of Troponin T Is A New Tropomyosin-Binding Site Modulating the Kinetics of Cardiac Muscle

Cao, T. — 2023-02-20

Evolved from duplication of a troponin I (TnI)-like ancestor gene, troponin T (TnT) and TnI are two subunits of the troponin complex that regulates the contraction and relaxation of striated muscles. Proteolytic deletion of the evolutionarily added N-terminal variable region of TnT in adaptation to acute myocardial stress restores a conformation like that of the C-terminal end segment of TnI, a tropomyosin (Tm)-binding and inhibitory structure, with an effect on reducing the contractile velocity of cardiac muscle to prolong ejection time and sustain the stroke volume. To investigate the underlying mechanism of this adaptive conditional function of TnT that is known to have two Tm-binding sites, our study localized a conformationally modulated new Tm-binding site in the highly conserved 14 amino acid C-terminal end segment of TnT. Localized surface plasmon resonance data showed that a hypertrophic cardiomyopathy mutation R278C within the C-terminal end segment of TnT alters the tropomyosin binding with direct responses to physiological concentrations of Ca2+. The functions are retained in the form of free peptide with an inhibitory regulatory effect on the contractile and relaxation kinetics of skinned cardiac muscle. In addition to revealing the underlying mechanisms of cTnT-ND adaptation and cardiac TnT C-terminal myopathic mutations, the new findings provide novel insights into the structure-function relationship of TnT in the kinetics of striated muscle contraction and relaxation with broad physiological and pathophysiological implications.

A cell state specific metabolic vulnerability to GPX4-dependent ferroptosis in glioblastoma

Banu, M. A. — 2023-02-23

Glioma cells hijack developmental transcriptional programs to control cell state. During neural development, lineage trajectories rely on specialized metabolic pathways. However, the link between tumor cell state and metabolic programs is poorly understood in glioma. Here we uncover a glioma cell state-specific metabolic liability that can be leveraged therapeutically. To model cell state diversity, we generated genetically engineered murine gliomas, induced by deletion of p53 alone (p53) or with constitutively active Notch signaling (N1IC), a pathway critical in controlling cellular fate. N1IC tumors harbored quiescent astrocyte-like transformed cell states while p53 tumors were predominantly comprised of proliferating progenitor-like cell states. N1IC cells exhibit distinct metabolic alterations, with mitochondrial uncoupling and increased ROS production rendering them more sensitive to inhibition of the lipid hydroperoxidase GPX4 and induction of ferroptosis. Importantly, treating patient-derived organotypic slices with a GPX4 inhibitor induced selective depletion of quiescent astrocyte-like glioma cell populations with similar metabolic profiles.

Myeloid Zfhx3 Deficiency Protects Against Hypercapnia-induced Suppression of Host Defense Against Influenza A Virus

Casalino-Matsuda, S. M. — 2023-03-01

Hypercapnia, elevation of the partial pressure of CO2 in blood and tissues, is a risk factor for mortality in patients with severe acute and chronic lung diseases. We previously showed that hypercapnia inhibits multiple macrophage and neutrophil antimicrobial functions, and that elevated CO2 increases the mortality of bacterial and viral pneumonia in mice. Here, we show that normoxic hypercapnia downregulates innate immune and antiviral gene programs in alveolar macrophages (AMOs). We also show that zinc finger homeobox 3 (Zfhx3), mammalian ortholog of zfh2, which mediates hypercapnic immune suppression in Drosophila, is expressed in mouse and human MOs. Deletion of Zfhx3 in the myeloid lineage blocked the suppressive effect of hypercapnia on immune gene expression in AMOs and decreased viral replication, inflammatory lung injury and mortality in hypercapnic mice infected with influenza A virus. Our results establish Zfhx3 as the first known mammalian mediator of CO2 effects on immune gene expression and lay the basis for future studies to identify therapeutic targets to interrupt hypercapnic immunosuppression in patients with advanced lung diseases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/530480v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@f6f907org.highwire.dtl.DTLVardef@577795org.highwire.dtl.DTLVardef@6c0068org.highwire.dtl.DTLVardef@8ebb72_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sleep Deprivation, Sleep Fragmentation and Social Jet Lag increase temperature preference in Drosophila

Roach, T. S. — 2023-03-02

Despite the fact that sleep deprivation substantially affects the way animals regulate their body temperature, the specific mechanisms behind this phenomenon are not well understood. In both mammals and flies, neural circuits regulating sleep and thermoregulation overlap, suggesting an interdependence that may be relevant for sleep function. To investigate this relationship further, we exposed flies to 12 h of sleep deprivation, or 48 h of sleep fragmentation and evaluated temperature preference in a thermal gradient. Flies exposed to 12 h of sleep deprivation chose warmer temperatures after sleep deprivation. Importantly, sleep fragmentation, which prevents flies from entering deeper stages of sleep, but does not activate sleep homeostatic mechanisms nor induce impairments in short-term memory also resulted in flies choosing warmer temperatures. To identify the underlying neuronal circuits, we used RNAi to knock down the receptor for Pigment dispersing factor, a peptide that influences circadian rhythms, temperature preference and sleep. Expressing UAS-PdfrRNAi in subsets of clock neurons prevented sleep fragmentation from increasing temperature preference. Finally, we evaluated temperature preference after flies had undergone a social jet lag protocol which is known to disrupt clock neurons. In this protocol, flies experience a 3 h light phase delay on Friday followed by a 3 h light advance on Sunday evening. Flies exposed to social jet lag exhibited an increase in temperature preference which persisted for several days. Our findings identify specific clock neurons that are modulated by sleep disruption to increase temperature preference. Moreover, our data indicate that temperature preference may be a more sensitive indicator of sleep disruption than learning and memory.

A Pillar and Perfusion Plate Platform for Robust Human Organoid Culture and Analysis

Kang, S.-Y. — 2023-03-13

Human organoids have potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo. This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, we overcome these challenges by engineering "microarray three-dimensional (3D) bioprinting" technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing and encapsulation techniques were demonstrated on a pillar plate, which was coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels were differentiated into liver and intestine organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

Gut microbiota and fecal short chain fatty acids differ with adiposity and country of origin: The METS-Microbiome Study

Ecklu-Mensah, G. — 2023-03-22

The relationship between the gut microbiota, short chain fatty acid (SCFA) metabolism, and obesity remains unclear due to conflicting reports from studies with limited statistical power. Additionally, this association has rarely been explored in large scale diverse populations. Here, we investigated associations between fecal microbial composition, predicted metabolic potential, SCFA concentrations, and obesity in a large (N = 1,934) adult cohort of African-origin spanning the epidemiologic transition, from Ghana, South Africa, Jamaica, Seychelles, and the United States (US). The greatest gut microbiota diversity and total fecal SCFA concentration was found in the Ghanaian population, while the lowest levels were found in the US population, respectively representing the lowest and the highest end of the epidemiologic transition spectrum. Country-specific bacterial taxa and predicted-functional pathways were observed, including an increased prevalence of Prevotella, Butyrivibrio, Weisella and Romboutsia in Ghana and South Africa, while Bacteroides and Parabacteroides were enriched in Jamaican and the US populations. Importantly, VANISH taxa, including Butyricicoccus and Succinivibrio, were significantly enriched in the Ghanaian cohort, reflecting the participants traditional lifestyles. Obesity was significantly associated with lower SCFA concentrations, a decrease in microbial richness, and dissimilarities in community composition, and reduction in the proportion of SCFA synthesizing bacteria including Oscillospira, Christensenella, Eubacterium, Alistipes, Clostridium and Odoribacter. Further, the predicted proportions of genes in the lipopolysaccharide (LPS) synthesis pathway were enriched in obese individuals, while genes associated with butyrate synthesis via the dominant pyruvate pathway were significantly reduced in obese individuals. Using machine learning, we identified features predictive of metabolic state and country of origin. Country of origin could accurately be predicted by the fecal microbiota (AUC = 0.97), whereas obesity could not be predicted as accurately (AUC = 0.65). Participant sex (AUC = 0.75), diabetes status (AUC = 0.63), hypertensive status (AUC = 0.65), and glucose status (AUC = 0.66) could all be predicted with different success. Interestingly, within country, the predictive accuracy of the microbiota for obesity was inversely correlated to the epidemiological transition, being greatest in Ghana (AUC = 0.57). Collectively, our findings reveal profound variation in the gut microbiota, inferred functional pathways, and SCFA synthesis as a function of country of origin. While obesity could be predicted accurately from the microbiota, the variation in accuracy in parallel with the epidemiological transition suggests that differences in the microbiota between obesity and non-obesity may be larger in low-to-middle countries compared to high-income countries. Further examination of independent study populations using multi-omic approaches will be necessary to determine the factors that drive this association.

Chromosomal Duplications of MurZ (MurA2) or MurA (MurA1), Amino Acid Substitutions in MurZ (MurA2), and Absence of KhpAB Obviate the Requirement for Protein Phosphorylation in Streptococcus pneumoniae D39

Tsui, H.-C. T. — 2023-03-27

GpsB links peptidoglycan synthases to other proteins that determine the shape of the respiratory pathogen Streptococcus pneumoniae (pneumococcus; Spn) and other low-GC Gram-positive bacteria. GpsB is also required for phosphorylation of proteins by the essential StkP(Spn) Ser/Thr protein kinase. Here we report three classes of frequently arising chromosomal duplications ({approx}21-176 genes) containing murZ (MurZ-family homolog of MurA) or murA that suppress {Delta}gpsB or {Delta}stkP. These duplications arose from three different repeated sequences and demonstrate the facility of pneumococcus to modulate gene dosage of numerous genes. Overproduction of MurZ or MurA alone or overexpression of MurZ caused by {Delta}khpAB mutations suppressed {Delta}gpsB or {Delta}stkP phenotypes to varying extents. {Delta}gpsB and {Delta}stkP were also suppressed by MurZ amino-acid changes distant from the active site, including one in commonly studied laboratory strains, and by truncation or deletion of the homolog of IreB(ReoM). Unlike in other Gram-positive bacteria, MurZ is predominant to MurA in pneumococcal cells. However, {Delta}gpsB and {Delta}stkP were not suppressed by {Delta}clpCP, which did not alter MurZ or MurA amounts. These results support a model in which regulation of MurZ and MurA activity, likely by IreB(Spn), is the only essential requirement for protein phosphorylation in exponentially growing D39 pneumococcal cells.

Five years later, with double the demographic data, naked mole-rat mortality rates continue to defy Gompertzian laws by not increasing with age

Ruby, J. G. — 2023-03-28

The naked mole-rat (Heterocephalus glaber) is a mouse-sized rodent species, notable for its eusociality and long lifespan. Previously, we reported that demographic aging, i.e., the exponential increase of mortality hazard that accompanies advancing age in mammals and other organisms, does not occur in naked mole-rats (Ruby et al, 2018). The demographic data supporting that conclusion had taken over three decades to accumulate, starting with the original rearing of H.glaber in captivity. In the five years following that study, we [~]doubled our quantity of demographic data. Here, we re-evaluated our prior conclusions in light of these new data and found them to be supported and indeed strengthened. We additionally provided insight into the social dynamics of captive H.glaber with data and analyses of body weight and colony size versus mortality. Finally, we provide a phylogenetically-proximal comparator in the form of lifespan data from our Damaraland mole-rat (Fukomys damarensis) colony and demographic meta-analysis of those data along with published data from Ansells mole-rat (Fukomys anselli). We found Fukomys mortality hazard to increase gradually with age, an observation with implications on the evolution of exceptional lifespan among mole-rats and the ecological factors that may have accompanied that evolution.

Increased Cerebral Serum Amyloid A2 and Parameters of Oxidation in Arylsulfatase B (N-Acetylgalactosamine-4-Sulfatase)-Null Mice

Tobacman, J. K. — 2023-04-03

IntroductionChondroitin sulfate and chondroitin sulfate proteoglycans have been associated with Alzheimers Disease (AD), and the impact of modified chondroitin sulfates is being investigated in several animal and cell-based models of AD. Published reports have shown the role of accumulation of chondroitin 4-sulfate and decline in Arylsulfatase B (ARSB; B-acetylgalactosamine-4-sulfatase) in other pathology, including nerve injury, traumatic brain injury, and spinal cord injury. However, the impact of ARSB deficiency on AD pathobiology has not been reported, although changes in ARSB were associated with AD in two prior reports. The enzyme ARSB removes 4-sulfate groups from the non-reducing end of chondroitin 4-sulfate and dermatan sulfate and is required for their degradation. When ARSB activity declines, these sulfated glycosaminoglycans accumulate, as in the inherited disorder Mucopolysaccharidosis VI. MethodsReports about chondroitin sulfate, chondroitin sulfate proteoglycans and chondroitin sulfatases in Alzheimers Disease were reviewed. Measurements of SAA2, iNOS, lipid peroxidation, chondroitin sulfate proteoglycan 4, and other parameters were performed in cortex and hippocampus from ARSB-null mice and controls by QRT-PCR, ELISA, and other standard assays. ResultsSAA2 mRNA expression and protein, CSPG4 mRNA, chondroitin 4-sulfate and i-NOS were increased significantly in ARSB-null mice. Measures of lipid peroxidation and redox state were significantly modified. DiscussionFindings indicate that decline in ARSB leads to changes in expression of parameters associated with AD in the hippocampus and cortex of the ARSB-deficient mouse. ConclusionsFurther investigation of the impact of decline in ARSB on the development of AD may provide a new approach to prevent and treat AD.

Metabolic signatures of cardiorenal dysfunction in plasma from sickle cell patients, as a function of therapeutic transfusion and hydroxyurea treatment

D'Alessandro, A. — 2023-04-06

Metabolomics studies in sickle cell disease (SCD) have been so far limited to tens of samples, owing to technical and experimental limitations. To overcome these limitations, we performed plasma metabolomics analyses on 596 samples from patients with sickle cell sickle cell disease (SCD) enrolled in the WALK-PHaSST study. Clinical covariates informed the biological interpretation of metabolomics data, including genotypes (hemoglobin SS, hemoglobin SC), history of recent transfusion (HbA%), response to hydroxyurea treatment (HbF%). We investigated metabolic correlates to the degree of hemolysis, cardiorenal function, as determined by tricuspid regurgitation velocity (TRV), estimated glomerular filtration rate (eGFR), and overall hazard ratio (unadjusted or adjusted by age). Recent transfusion events or hydroxyurea treatment were associated with elevation in plasma free fatty acids and decreases in acyl-carnitines, urate, kynurenine, indoles, carboxylic acids, and glycine- or taurine-conjugated bile acids. High levels of these metabolites, along with low levels of plasma S1P and L-arginine were identified as top markers of hemolysis, cardiorenal function (TRV, eGFR), and overall hazard ratio. We thus uploaded all omics and clinical data on a novel online portal that we used to identify a potential mechanism of dysregulated red cell S1P synthesis and export as a contributor to the more severe clinical manifestations in patients with the SS genotype compared to SC. In conclusion, plasma metabolic signatures - including low S1P, arginine and elevated kynurenine, acyl-carnitines and bile acids - are associated with clinical manifestation and therapeutic efficacy in SCD patients, suggesting new avenues for metabolic interventions in this patient population.

Limited Diffusion of Scientific Knowledge Forecasts Collapse

Kang, D. — 2023-04-17

Market bubbles emerge when asset prices are driven unsustainably higher than asset values and shifts in belief burst them. We demonstrate the same phenomenon for biomedical knowledge when promising research receives inflated attention. We predict deflationary events by developing a diffusion index that captures whether research areas have been amplified within social and scientific bubbles or have diffused and become evaluated more broadly. We illustrate our diffusion approach contrasting the trajectories of cardiac stem cell research and cancer immunotherapy. We then trace the diffusion of unique 28,504 subfields in biomedicine comprising nearly 1.9M papers and more than 80M citations and demonstrate that limited diffusion of biomedical knowledge anticipates abrupt decreases in popularity. Our analysis emphasizes that restricted diffusion, implying a socio-epistemic bubble, leads to dramatic collapses in relevance and attention accorded to scientific knowledge.

Growth Hormone Accelerates Recovery From Acetaminophen-Induced Murine Liver Injury

Everton, E. — 2023-04-18

Background and AimsAcetaminophen (APAP) overdose is the leading cause of acute liver failure, with one available treatment, N-acetyl cysteine (NAC). Yet, NAC effectiveness diminishes about ten hours after APAP overdose, urging for therapeutic alternatives. This study addresses this need by deciphering a mechanism of sexual dimorphism in APAP-induced liver injury, and leveraging it to accelerate liver recovery via growth hormone (GH) treatment. GH secretory patterns, pulsatile in males and near-continuous in females, determine the sex bias in many liver metabolic functions. Here, we aim to establish GH as a novel therapy to treat APAP hepatotoxicity. Approach and ResultsOur results demonstrate sex-dependent APAP toxicity, with females showing reduced liver cell death and faster recovery than males. Single-cell RNA sequencing analyses reveal that female hepatocytes have significantly greater levels of GH receptor expression and GH pathway activation compared to males. In harnessing this female-specific advantage, we demonstrate that a single injection of recombinant human GH protein accelerates liver recovery, promotes survival in males following sub-lethal dose of APAP, and is superior to standard-of-care NAC. Alternatively, slow-release delivery of human GH via the safe nonintegrative lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP), a technology validated by widely used COVID-19 vaccines, rescues males from APAP-induced death that otherwise occurred in control mRNA-LNP-treated mice. ConclusionsOur study demonstrates a sexually dimorphic liver repair advantage in females following APAP overdose, leveraged by establishing GH as an alternative treatment, delivered either as recombinant protein or mRNA-LNP, to potentially prevent liver failure and liver transplant in APAP-overdosed patients.

In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1-mutant lung cancer

Deng, J. — 2023-04-20

LKB1/STK11 is a serine/threonine kinase that plays a major role in controlling cell metabolism, resulting in potential therapeutic vulnerabilities in LKB1-mutant cancers. Here, we identify the NAD+ degrading ectoenzyme, CD38, as a new target in LKB1-mutant NSCLC. Metabolic profiling of genetically engineered mouse models (GEMMs) revealed that LKB1 mutant lung cancers have a striking increase in ADP-ribose, a breakdown product of the critical redox co-factor, NAD+. Surprisingly, compared with other genetic subsets, murine and human LKB1-mutant NSCLC show marked overexpression of the NAD+-catabolizing ectoenzyme, CD38 on the surface of tumor cells. Loss of LKB1 or inactivation of Salt-Inducible Kinases (SIKs)--key downstream effectors of LKB1-- induces CD38 transcription induction via a CREB binding site in the CD38 promoter. Treatment with the FDA-approved anti-CD38 antibody, daratumumab, inhibited growth of LKB1-mutant NSCLC xenografts. Together, these results reveal CD38 as a promising therapeutic target in patients with LKB1 mutant lung cancer. SIGNIFICANCELoss-of-function mutations in the LKB1 tumor suppressor of lung adenocarcinoma patients and are associated with resistance to current treatments. Our study identified CD38 as a potential therapeutic target that is highly overexpressed in this specific subtype of cancer, associated with a shift in NAD homeostasis.

Myosin Light Chain Dephosphorylation by PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation

McCauley, M. D. — 2023-04-20

BackgroundAtrial fibrillation (AF), the most common sustained cardiac arrhythmia, increases thromboembolic stroke risk five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C, the PP1 regulatory subunit targeting atrial myosin light chain 2 (MLC2a), causes hypophosphorylation of MLC2a and results in atrial hypocontractility. MethodsRight atrial appendage tissues were isolated from human AF patients versus sinus rhythm (SR) controls. Western blots, co-immunoprecipitation, and phosphorylation studies were performed to examine how the PP1c-PPP1R12C interaction causes MLC2a de-phosphorylation. In vitro studies of pharmacologic MRCK inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with EP studies. ResultsIn human patients with AF, PPP1R12C expression was increased two-fold versus SR controls (P=2.0x10-2, n=12,12 in each group) with > 40% reduction in MLC2a phosphorylation (P=1.4x10-6, n=12,12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (P=2.9x10-2 and 6.7x10-3 respectively, n=8,8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Lenti-12C mice demonstrated a 150% increase in LA size versus controls (P=5.0x10-6, n=12,8,12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in Lenti-12C mice was significantly higher than controls (P=1.8x10-2 and 4.1x10-2 respectively, n= 6,6,5). ConclusionsAF patients exhibit increased levels of PPP1R12C protein compared to controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

Combined inactivation of RB and Hippo pathways converts differentiating photoreceptors into eye progenitor cells through derepression of homothorax

Rader, A. E. — 2023-04-24

The RB and Hippo pathways interact to regulate cell proliferation and differentiation. However, their mechanism of interaction is not fully understood. Drosophila photoreceptors with inactivated RB and Hippo pathways specify normally but fail to maintain neuronal identity and dedifferentiate. We performed single-cell RNA-sequencing to elucidate the cause of dedifferentiation and the fate of these cells. We find that dedifferentiated cells adopt a progenitor-like fate due to inappropriate activation of the retinal differentiation suppressor homothorax (hth) by Yki/Sd. This results in activation of the Yki/Hth transcriptional program, driving photoreceptor dedifferentiation. We show that Rbf physically interacts with Yki which, together with the GAGA factor, inhibits hth expression. Thus, RB and Hippo pathways cooperate to maintain photoreceptor differentiation by preventing inappropriate expression of hth in differentiating photoreceptors. Our work accentuates the importance of both RB and Hippo pathway activity for maintaining the state of terminal differentiation.

Tension sensing by FAK governs nuclear mechanotransduction, endothelial transcriptome and fate

Akhter, M. Z. — 2023-04-26

Vascular endothelium forms a restrictive barrier to defend the underlying tissue against uncontrolled influx of circulating protein and immune cells. Mechanisms that mediate the transition from restrictive to leaky endothelium, a hallmark of tissue injury exemplified by acute lung injury (ALI), remain elusive. Using endothelial cell (EC)-Fak-/-mice, we show that FAK sensing and transmission of mechanical tension to the EC nucleus governs cell fate. In FAK- deleted EC, increased EC tension induced by Rho kinase caused tyrosine phosphorylation of nuclear envelope protein, emerin at Y74/Y95, and its localization in a nuclear cap. Activated emerin stimulated DNMT3a activity and methylation of the KLF2 promoter, impairing the restrictive EC transcriptome, including S1PR1. Inhibiting emerin phosphorylation or DNMT3a activity enabled KLF2 transcription of S1PR1, rescuing the restrictive EC phenotype in EC-Fak-/- lungs. Thus, FAK sensing of tension transmission to the nucleus is crucial for maintaining a restrictive EC fate and lung homeostasis.

Diet-induced obesity dysregulates chromatin oxygen sensing regulating efferocytosis in macrophages

Takahashi, K. — 2023-05-14

Macrophages are plastic cell populations that normally adapt to their environment. Cellular adaptation to hypoxia occurs through transcription factors including hypoxia-inducible factors, and hypoxia-inducible transcriptions are further regulated by chromatin response through histone modification including histone methylation. However, the role of histone methylation in the hypoxia response of macrophages is not well understood. As obesity is associated with dysregulated macrophage functions, we investigated whether hypoxia response is cell-intrinsically dysregulated in macrophages in obesity. In mouse bone marrow-derived macrophages (BMDMs), immunoblotting revealed that 1% hypoxia rapidly increases the global levels of histone 3 methylations. We found that hypoxia-induction of histone 3-lysine 4 tri-methylation (H3K4me3) is specifically inhibited in BMDMs from mice fed a high-fat diet (HFD-BMDMs) compared to BMDMs from mice fed a normal diet (ND-BMDMs). Multi-omics approach with ChIP-seq and RNA-seq identified that glycolysis-related pathways and genes including Aldoa are upregulated after prolonged hypoxia along with upregulated H3K4me3 in ND-BMDMs. In contrast, no pathway is associated with hypoxia-upregulated H3K4me3 peaks in HFD-BMDMs and hypoxia-induced Aldoa expression is decreased in HFD-BMDMs, suggesting both the extent and the genome location of H3K4me3 response to hypoxia is dysregulated in obesity. Consistently, lactate accumulation and induction of histone lactylation under hypoxia are reduced in HFD-BMDMs. Furthermore, HFD-BMDMs exhibited decreased dying cell clearance under hypoxia due to the reduced capacity of anaerobic glycolysis. Competitive bone marrow transplantation of hematopoietic stem cells (HSCs) shows that HFD-induced long-term memory reflects the impaired dying cell clearance in differentiated BMDMs, which is rescued by inhibiting oxidative stress in HSCs. In summary, chromatin response to hypoxia associated with H3K4me3 enrichment governs transcriptions for anaerobic glycolysis and dying cell clearance under hypoxia. Obesity dysregulates the extent and the genome location of H3K4me3 enrichment, glycolysis, and dying cell clearance of BMDMs under hypoxia, which is initiated in HSPCs via oxidative stress.

DNA damage primes hematopoietic stem cells for direct megakaryopoiesis

Garyn, C. M. — 2023-05-15

Hematopoietic stem cells (HSCs) reside in the bone marrow (BM), can self-renew, and generate all cells of the hematopoietic system.1 Most hematopoietic lineages arise through successive, increasingly lineage-committed progenitors. In contrast, megakaryocytes (MKs), hyperploid cells that generate platelets essential to hemostasis, can derive rapidly and directly from HSCs.2 The underlying mechanism is unknown however. Here we show that DNA damage and subsequent arrest in the G2 phase of the cell cycle rapidly induce MK commitment specifically in HSCs, but not in progenitors, through an initially predominantly post-transcriptional mechanism. Cycling HSCs show extensive replication-induced DNA damage associated with uracil misincorporation in vivo and in vitro. Consistent with this notion, thymidine attenuated DNA damage, rescued HSC maintenance and reduced the generation of CD41+ MK-committed HSCs in vitro. Similarly, overexpression of the dUTP-scavenging enzyme, dUTPase, enhanced in vitro maintenance of HSCs. We conclude that a DNA damage response drives direct megakaryopoiesis and that replication stress-induced direct megakaryopoiesis, at least in part caused by uracil misincorporation, is a barrier to HSC maintenance in vitro. DNA damage-induced direct megakaryopoiesis may allow rapid generation of a lineage essential to immediate organismal survival, while simultaneously removing damaged HSCs and potentially avoiding malignant transformation of self-renewing stem cells.

Transcriptomic Evaluation of Stress Vulnerability Network using Single Cell RNA-Seq in mouse Prefrontal Cortex

Hing, B. — 2023-05-14

Increased vulnerability to stress is a major risk factor for the manifestation of several mood disorders, including major depressive disorder (MDD). Despite the status of MDD as a significant donor to global disability, the complex integration of genetic and environmental factors that contribute to the behavioral display of such disorders has made a thorough understanding of related etiology elusive. Recent developments suggest that a brain-wide network approach is needed, taking into account the complex interplay of cell types spanning multiple brain regions. Single cell RNA-sequencing technologies can provide transcriptomic profiling at the single-cell level across heterogenous samples. Furthermore, we have previously used local field potential oscillations and machine learning to identify an electrical brain network that is indicative of a predisposed vulnerability state. Thus, this study combined single cell RNA-sequencing (scRNA-Seq) with electrical brain network measures of the stress-vulnerable state, providing a unique opportunity to access the relationship between stress network activity and transcriptomic changes within individual cell types. We found especially high numbers of differentially expressed genes between animals with high and low stress vulnerability brain network activity in astrocytes and glutamatergic neurons but we estimated that vulnerability network activity depends most on GABAergic neurons. High vulnerability network activity included upregulation of microglia and mitochondrial and metabolic pathways, while lower vulnerability involved synaptic regulation. Genes that were differentially regulated with vulnerability network activity significantly overlapped with genes identified as having significant SNPs by human GWAS for depression. Taken together, these data provide the gene expression architecture of a previously uncharacterized stress vulnerability brain state, enabling new understanding and intervention of predisposition to stress susceptibility. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/540705v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1ebe036org.highwire.dtl.DTLVardef@cd2131org.highwire.dtl.DTLVardef@13e3519org.highwire.dtl.DTLVardef@1100e62_HPS_FORMAT_FIGEXP M_FIG C_FIG

Changes in Active Site Loop Conformation Relate to the Transition toward a Novel Enzymatic Activity

Jacquet, P. — 2023-05-22

Enzymatic promiscuity, the ability of enzymes to catalyze multiple, distinct chemical reactions, has been well documented and is hypothesized to be a major driver for the emergence of new enzymatic functions. Yet, the molecular mechanisms involved in the transition from one activity to another remain debated and elusive. Here, we evaluated the redesign of the active site binding cleft of the lactonase SsoPox using structure-based design and combinatorial libraries. We created variants with largely improved catalytic abilities against phosphotriesters, the best ones being > 1,000-fold better compared to the wild-type enzyme. The observed shifts in activity specificity are large, [~]1,000,000-fold and beyond, since some variants completely lost their initial activity. The selected combinations of mutations have considerably reshaped the active site cavity via side chain changes but mostly through large rearrangements of the active site loops, as revealed by a suite of crystal structures. This suggests that specific active site loop configuration is critical to the lactonase activity. Interestingly, analysis of high-resolution structures hints at the potential role of conformational sampling and its directionality in defining an enzyme activity profile.

The Notch-Pdgfr beta axis suppresses brown adipocyte progenitor differentiation in early postnatal mice

Shi, Z. — 2023-05-24

De novo brown adipogenesis holds potential in combating the epidemics of obesity and diabetes. However, the identity of brown adipocyte progenitor cells (APCs) and their regulation have not been extensively studied. Here through in vivo lineage tracing, we observed that PDGFR{beta}+ pericytes give rise to developmental brown adipocytes, but not to those in adult homeostasis. In contrast, TBX18+ pericytes contribute to brown adipogenesis throughout both developmental and adult stages, though in a depot-specific manner. Mechanistically, Notch inhibition in PDGFR{beta}+ pericytes promotes brown adipogenesis through the downregulation of PDGFR{beta}. Furthermore, inhibition of Notch signaling in PDGFR{beta}+ pericytes mitigates HFHS (high-fat, high-sucrose) induced glucose and metabolic impairment in both developmental and adult stages. Collectively, these findings show that the Notch/PDGFR{beta} axis negatively regulates developmental brown adipogenesis, and its repression promotes brown adipose tissue expansion and improves metabolic health. HighlightsO_LIPDGFR{beta}+ pericytes act as an essential developmental brown APC. C_LIO_LITBX18+ pericytes contribute to brown adipogenesis in a depot-specific manner. C_LIO_LIInhibiting Notch-Pdgfr{beta} axis promotes brown APC adipogenesis. C_LIO_LIEnhanced postnatal brown adipogenesis improves metabolic health in adult stage. C_LI

A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

Dower, W. J. — 2023-05-24

Interleukin (IL)-7 is broadly active on T-cell populations, and modified versions have been clinically evaluated for a variety of therapeutic applications, including cancer, lymphopenia and infectious diseases; and found to be relatively well-tolerated and biologically active. Here we describe novel IL-7R agonists that are unrelated in structure to IL-7, bind to the receptor subunits differently from IL-7, but closely emulate IL-7 biology. The small size, low structural complexity, and the natural amino acid composition of the pharmacologically active peptide MDK1472 allows facile incorporation into protein structures, such as the IgG2-Fc fusion MDK-703. This molecule possesses properties potentially better suited to therapeutic applications than native IL-7 or its derivatives. We compared these compounds with IL-7 for immune cell selectivity, induction of IL-7R signaling, receptor-mediated internalization, proliferation, and generation of immune cell phenotypes in human and non-human primate (NHP) peripheral blood cells in vitro; and found them to be similar in biological activity to IL-7. In cynomolgus macaques, MDK-703 exhibits a circulating half-life of 46 hr, and produces sustained T-cell expansion characteristic of IL-7 treatment. In the huCD34+-engrafted NSG mouse model of the human immune system, MDK-703 induces an immune cell profile very similar to that generated by IL-7-derived compounds; including the pronounced expansion of memory T-cells, particularly the population of stem-like memory T-cells (Tscm), which may be important for anti-tumor activities reported with IL-7 treatment. Clinical administration of IL-7 and modified variants has been reported to induce anti-drug antibodies (ADAs), including IL-7 neutralizing antibodies. The novel peptide agonist reported here scores very low in predicted immunogenicity, and because the peptide lacks sequence similarity with IL-7, the problematic immunogenic neutralization of endogenous cytokine should not occur.

Exogenous Recombinant N-Acetylgalactosamine-4-Sulfatase (Arylsulfatase B; ARSB) Inhibits Progression of B16F10 Cutaneous Melanomas and Modulates Cell Signaling

Bhattacharyya, S. — 2023-05-31

In the syngeneic, subcutaneous B16F10 mouse model of malignant melanoma, treatment with exogenous ARSB markedly reduced tumor size and extended survival. In vivo experiments showed that local treatment with exogenous N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) led to reduced tumor growth over time (p<0.0001) and improved the probability of survival up to 21 days (p=0.0391). Tumor tissue from the treated mice had lower chondroitin 4-sulfate (C4S) content and lower sulfotransferase activity. The free galectin-3 declined, and the SHP2 activity increased, due to altered binding with chondroitin 4-sulfate. These changes induced effects on transcription, which were mediated by Sp1, phospho-ERK1/2, and phospho-p38 MAPK. Reduced mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4), chondroitin sulfotransferase 15 (N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase), and matrix metalloproteinases 2 and 9 resulted. Experiments in the human melanoma cell line A375 demonstrated similar responses to exogenous ARSB as in the tumors, and inverse effects followed RNA silencing. ARSB, which removes the 4-sulfate group at the non-reducing end of C4S, acts as a tumor suppressor, and treatment with exogenous ARSB impacts on vital cell signaling and reduces the expression of critical genes associated with melanoma progression. HighlightsExogenous ARSB reduced tumor size and increased survival Chondroitin 4-sulfate increased, leading to increased free galectin-3 mRNA expression of CSPG4 and CHST15 declined following ARSB treatment mRNA expression of MMP9 and pro-MMP2 declined following ARSB treatment Active SHP2 increased, leading to declines in phospho-ERK1/2 and phospho-p38 MAPK

Expression of RcrB confers resistance to hypochlorous acid in uropathogenic Escherichia coli

Crompton, M. E. — 2023-06-01

To eradicate bacterial pathogens, neutrophils are recruited to the sites of infection, where they engulf and kill microbes through the production of reactive oxygen and chlorine species (ROS/RCS). The most prominent RCS is antimicrobial oxidant hypochlorous acid (HOCl), which rapidly reacts with various amino acids side chains, including those containing sulfur and primary/tertiary amines, causing significant macromolecular damage. Pathogens like uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections (UTIs), have developed sophisticated defense systems to protect themselves from HOCl. We recently identified the RcrR regulon as a novel HOCl defense strategy in UPEC. The regulon is controlled by the HOCl-sensing transcriptional repressor RcrR, which is oxidatively inactivated by HOCl resulting in the expression of its target genes, including rcrB. rcrB encodes the putative membrane protein RcrB, deletion of which substantially increases UPECs susceptibility to HOCl. However, many questions regarding RcrBs role remain open including whether (i) the proteins mode of action requires additional help, (ii) rcrARB expression is induced by physiologically relevant oxidants other than HOCl, and (iii) expression of this defense system is limited to specific media and/or cultivation conditions. Here, we provide evidence that RcrB expression is sufficient to E. colis protection from HOCl and induced by and protects from several RCS but not from ROS. RcrB plays a protective role for RCS-stressed planktonic cells under various growth and cultivation conditions but appears to be irrelevant for UPECs biofilm formation. IMPORTANCEBacterial infections pose an increasing threat to human health exacerbating the demand for alternative treatment options. UPEC, the most common etiological agent of urinary tract infections (UTIs), are confronted by neutrophilic attacks in the bladder, and must therefore be well equipped with powerful defense systems to fend off the toxic effects of RCS. How UPEC deal with the negative consequences of the oxidative burst in the neutrophil phagosome remains unclear. Our study sheds light on the requirements for the expression and protective effects of RcrB, which we recently identified as UPECs most potent defense system towards HOCl-stress and phagocytosis. Thus, this novel HOCl-stress defense system could potentially serve as an attractive drug target to increase the bodys own capacity to fight UTIs.

Mechanisms of Pathogenicity of Hypertrophic Cardiomyopathy-Associated Troponin T (TNNT2) Variant R278C+/- During Development

Shafaattalab, S. — 2023-06-07

Hypertrophic cardiomyopathy (HCM) is one of the most common heritable cardiovascular diseases and variants of TNNT2 (cardiac troponin T) are linked to increased risk of sudden cardiac arrest despite causing limited hypertrophy. In this study, a TNNT2 variant, R278C+/-, was generated in both human cardiac recombinant/reconstituted thin filaments (hcRTF) and human-induced pluripotent stem cells (hiPSCs) to investigate the mechanisms by which the R278C+/- variant affects cardiomyocytes at the proteomic and functional levels. The results of proteomics analysis showed a significant upregulation of markers of cardiac hypertrophy and remodeling in R278C+/- vs. the isogenic control. Functional measurements showed that R278C+/- variant enhances the myofilament sensitivity to Ca2+, increases the kinetics of contraction, and causes arrhythmia at frequencies >75 bpm. This study uniquely shows the profound impact of the TNNT2 R278C+/- variant on the cardiomyocyte proteomic profile, cardiac electrical and contractile function in the early stages of cardiac development. Translational PerspectiveHypertrophic cardiomyopathy (HCM) is the leading known cause of sudden cardiac arrest in the young. Thin-variant associated HCM variants make up to 15% of familial HCM yet their molecular mechanisms remain less clear relative to thick filament variants. Here, we employ computational modeling, human cardiac recombinant/reconstituted thin filaments (hcRTF), and hiPSC-CMs to study the thin filament TNNT2 R278C+/- variant, revealing its extensive pathogenicity and potential mechanisms by which it can lead to HCM and sudden death. Mavacamten, the recently FDA-approved treatment, was effective in alleviating contractile dysfunction in TNNT2 R278C+/- hiPSC-CMs, positing it as a potential therapy for thin filament HCM. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=117 HEIGHT=200 SRC="FIGDIR/small/542948v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@102a8b0org.highwire.dtl.DTLVardef@190e356org.highwire.dtl.DTLVardef@13d3f9aorg.highwire.dtl.DTLVardef@1a9c33_HPS_FORMAT_FIGEXP M_FIG C_FIG

Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

Reed, E. B. — 2023-06-07

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-beta (TGF-{beta}) is one of the most established drivers of fibrotic processes. TGF-{beta} promotes transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-{beta} robustly upregulates the expression of the calcium-activated chloride channel Anoctamin-1 (ANO1) in human lung fibroblasts (HLF) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle alpha-actin (SMA)-positive myofibroblasts. TGF-{beta}-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1 and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate pro-fibrotic TGF-{beta} signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-{beta} treatment of HLF results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-{beta}-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that (i) ANO1 is a TGF-{beta}-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-{beta}; and (ii) ANO1 mediates TGF-{beta}-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein, but independent of WNK1 kinase activity. NEW & NOTEWORTHYThis study describes a novel mechanism of differentiation of human lung fibroblasts (HLF) to myofibroblasts - the key process in the pathogenesis of pulmonary fibrosis. TGF-{beta} drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive With-No-Lysine (K) kinase (WNK1) to activate pro-fibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.

Neuropathy target esterase activity predicts retinopathy among PNPLA6 disorders

Liu, J. — 2023-06-11

Biallelic pathogenic variants in the PNPLA6 gene cause a broad spectrum of disorders leading to gait disturbance, visual impairment, anterior hypopituitarism, and hair anomalies. PNPLA6 encodes Neuropathy target esterase (NTE), yet the role of NTE dysfunction on affected tissues in the large spectrum of associated disease remains unclear. We present a clinical meta-analysis of a novel cohort of 23 new patients along with 95 reported individuals with PNPLA6 variants that implicate missense variants as a driver of disease pathogenesis. Measuring esterase activity of 46 disease-associated and 20 common variants observed across PNPLA6-associated clinical diagnoses unambiguously reclassified 10 variants as likely pathogenic and 36 variants as pathogenic, establishing a robust functional assay for classifying PNPLA6 variants of unknown significance. Estimating the overall NTE activity of affected individuals revealed a striking inverse relationship between NTE activity and the presence of retinopathy and endocrinopathy. This phenomenon was recaptured in vivo in an allelic mouse series, where a similar NTE threshold for retinopathy exists. Thus, PNPLA6 disorders, previously considered allelic, are a continuous spectrum of pleiotropic phenotypes defined by an NTE genotype:activity:phenotype relationship. This relationship and the generation of a preclinical animal model pave the way for therapeutic trials, using NTE as a biomarker.

Engineered CD47 protects T cells for enhanced antitumor immunity

Yamada-Hunter, S. A. — 2023-06-22

Adoptively transferred T cells and agents designed to block the CD47/SIRP axis are promising antitumor therapeutics, which activate distinct arms of the immune system. We administered anti-CD47 (CD47) with adoptively transferred T cells with the goal of enhancing antitumor efficacy but observed rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors, which blunted therapeutic benefit. CD47 mediated CAR T clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered a CD47 variant (47E) that engaged SIRP and provided a "dont-eat-me" signal that was not blocked by CD47 antibodies. TCR or CAR T cells expressing 47E were resistant to clearance by macrophages following CD47, and mediated significant, sustained macrophage recruitment into the TME. Although many of the recruited macrophages manifested an M2-like profile, the combined therapy resulted in synergistic enhancement in antitumor efficacy. This work identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T cell directed therapeutics with those designed to activate macrophages. It further delivers a therapeutic approach capable of simultaneously harnessing the antitumor effects of T cells and macrophages that manifests markedly enhanced potency against solid tumors.

Neural circuits for fast Poisson compressed sensing in the olfactory bulb

Zavatone-Veth, J. A. — 2023-06-24

Within a single sniff, the mammalian olfactory system can decode the identity and concentration of odorants wafted on turbulent plumes of air. Yet, it must do so given access only to the noisy, dimensionally-reduced representation of the odor world provided by olfactory receptor neurons. As a result, the olfactory system must solve a compressed sensing problem, relying on the fact that only a handful of the millions of possible odorants are present in a given scene. Inspired by this principle, past works have proposed normative compressed sensing models for olfactory decoding. However, these models have not captured the unique anatomy and physiology of the olfactory bulb, nor have they shown that sensing can be achieved within the 100-millisecond timescale of a single sniff. Here, we propose a rate-based Poisson compressed sensing circuit model for the olfactory bulb. This model maps onto the neuron classes of the olfactory bulb, and recapitulates salient features of their connectivity and physiology. For circuit sizes comparable to the human olfactory bulb, we show that this model can accurately detect tens of odors within the timescale of a single sniff. We also show that this model can perform Bayesian posterior sampling for accurate uncertainty estimation. Fast inference is possible only if the geometry of the neural code is chosen to match receptor properties, yielding a distributed neural code that is not axis-aligned to individual odor identities. Our results illustrate how normative modeling can help us map function onto specific neural circuits to generate new hypotheses.

Endothelial Knockdown of the Tumor Suppressor, WWOX, Increases Inflammation in Ventilator-Induced Lung Injury

Zeng, Z. — 2023-06-30

BackgroundChronic cigarette smoke exposure downregulates lung expression of WWOX, an ARDS relevant tumor suppressor. Prior work has revealed a barrier protective function of WWOX during infectious models of ARDS. Proteomic analysis of WWOX-silenced lung endothelial cells suggest involvement of WWOX in protection against mechanical stretch-induced inflammation. MethodsProtein lysates from WWOX-silenced endothelial cells (ECs) were analyzed using tandem mass tag mass spectrometry (TMT-MS) to determine the differential expression status of the proteome compared to wild type ECs. WWOX-silenced ECs as well as those isolated from endothelial Wwox knockout (EC Wwox KO) mice were subjected to cyclic stretch (18% elongation, 0.5 Hz, 4 hours). Cellular lysates and media supernatant were harvested for assays of cellular signaling, protein expression, and cytokine release. Dual silencing of WWOX and zyxin was achieved to determine the role of zyxin upregulation in IL-8 production following mechanical stretch and during WWOX knockdown. Control and EC Wwox KO mice were subjected to high tidal volume ventilation (VILI, 40ml/kg, 65 breath/min, 4hours). Bronchoalveolar lavage fluid and mouse lung tissue were harvested for cellular signaling, cytokine secretion, and histologic assays. ResultsTMT-MS revealed upregulation of zyxin expression during WWOX knockdown which predicted a heightened inflammatory response to mechanical stretch. WWOX-silenced ECs and ECs isolated from EC Wwox mice displayed significantly increased cyclic stretch-mediated secretion of various cytokines (IL-6, KC/IL-8, IL-1{beta}, and MCP-1) relative to controls. This was associated with increased ERK and JNK phosphorylation but decreased p38 MAPK phosphorylation. EC Wwox KO mice subjected to VILI sustained a greater degree of injury than corresponding controls. Silencing of zyxin during WWOX knockdown abrogated stretch-induced increases in IL-8 secretion. ConclusionLoss of WWOX function in ECs is associated with a heightened inflammatory response during mechanical stretch that is associated with increased MAPK phosphorylation and appears to be dependent on upregulation of zyxin.

Grand Biological Universe: Genome space geometry unravels looking for a single metric is likely to be futile in evolution

Sun, N. — 2023-07-08

Understanding the differences between genomic sequences of different lives is crucial for biological classification and phylogeny. Here, we downloaded all the reliable sequences of the seven kingdoms and determined the dimensions of the genome space embedded in the Euclidean space, along with the corresponding Natural Metrics. The concept of the Grand Biological Universe is further proposed. In the grand universe, the convex hulls formed by the universes of seven kingdoms are mutually disjoint, and the convex hulls formed by different biological groups within each kingdom are mutually disjoint. This study provides a novel geometric perspective for studying molecular biology and also offers an accurate way for large-scale sequence comparison in a real-time manner. Most importantly, this study shows that, due to the space-time distortion in the biological genome space similar to Einsteins theory, it is futile to look for a single metric to measure different biological universes, as previous studies have done.

Validation of human telomere length trans-ancestry meta-analysis association signals identifies POP5 and KBTBD6 as novel human telomere length regulation genes

Keener, R. — 2023-07-14

Telomere length genome-wide association studies (GWAS) have become well-powered to detect novel genes in telomere length regulation. However, no prior work has validated these putative novel genes to confirm the contribution of GWAS loci to telomere length regulation. We conducted a trans-ancestry meta-analysis of 211,369 individuals. Through enrichment analyses of chromatin state and cell-type heritability we identified blood and immune cells as the most relevant cell type to examine telomere length association signals. We validated specific GWAS associations by overexpressing KBTBD6, a component of an E3 ubiquitin ligase complex, and POP5, a component of the Ribonuclease P/MRP complex, and demonstrating that both lengthened telomeres as predicted by our statistical analyses. CRISPR/Cas9 deletion of the predicted causal regions of these association peaks in K562 immortalized blood cells reduced expression of these genes, demonstrating that these loci are related to transcriptional regulation of KBTBD6 and POP5, respectively. Together our results demonstrate the utility of telomere length GWAS in the identification of novel telomere length regulation mechanisms and highlight the importance of the proteasome-ubiquitin pathway in telomere length regulation.

Prostaglandins as Candidate Ligands for a Per-ARNT-Sim (PAS) Domain of Steroid Receptor Coactivator 1 (SRC1)

Daffern, N. — 2023-07-13

Steroid receptor coactivators (SRCs) comprise a family of three paralogous proteins commonly recruited by eukaryotic transcription factors. Each SRC harbors two tandem Per-ARNT-Sim (PAS) domains that are broadly distributed that bind small molecules and regulate interactions. Using computational docking, solution NMR, mass spectrometry, and molecular dynamics simulations, we show that the SRC1 PAS-B domain can bind to certain prostaglandins (PGs) either non-covalently to a surface that overlaps with the site used to engage transcription factors or covalently to a single, specific, conserved cysteine residue next to a solvent accessible hydrophobic pocket. This pocket is in proximity to the canonical transcription factor binding site, but on the opposite side of the domain, suggesting a potential mode of regulating transcriptional activator-coactivator interactions.

Microstructure-Based Modeling of Primary Cilia Mechanics

Mostafazadeh, N. — 2023-07-15

A primary cilium, made of nine microtubule doublets enclosed in a cilium membrane, is a mechanosensing organelle that bends under an external mechanical load and sends an intracellular signal through transmembrane proteins activated by cilium bending. The nine microtubule doublets are the main load-bearing structural component, while the transmembrane proteins on the cilium membrane are the main sensing component. No distinction was made between these two components in all existing models, where the stress calculated from the structural component (nine microtubule doublets) was used to explain the sensing location, which may be totally misleading. For the first time, we developed a microstructure-based primary cilium model by considering these two components separately. First, we refined the analytical solution of bending an orthotropic cylindrical shell for individual microtubule, and obtained excellent agreement between finite element simulations and the theoretical predictions of a microtubule bending as a validation of the structural component in the model. Second, by integrating the cilium membrane with nine microtubule doublets, we found that the microtubule doublets may twist significantly as the whole cilium bends. Third, besides being cilium-length-dependent, we found the mechanical properties of the cilium are also highly deformation-dependent. More important, we found that the cilium membrane near the base is not under pure in-plane tension or compression as previously thought, but has significant local bending stress. This challenges the traditional model of cilium mechanosensing, indicating that transmembrane proteins may be activated more by membrane curvature than membrane stretching. Finally, we incorporated imaging data of primary cilia into our microstructure-based cilium model, and found that comparing to the ideal model with uniform microtubule length, the imaging-informed model shows the nine microtubule doublets interact more evenly with the cilium membrane, and their contact locations can cause even higher bending curvature in the cilium membrane than near the base. SIGNIFICANCEFactors regulating the mechanical response of a primary cilium to fluid flow remain unclear. Modeling the microtubule doublet as a composite of two orthotropic shells and the ciliary axoneme as an elastic shell enclosing nine such microtubule doublets, we found that the length distribution of microtubule doublets (inferred from cryogenic electron tomography images) is the primary determining factor in the bending stiffness of primary cilia, rather than just the ciliary length. This implies ciliary-associated transmembrane proteins may be activated by membrane curvature changes rather than just membrane stretching. These insights challenge the traditional view of ciliary mechanosensation and expands our understanding of the different ways in which cells perceive and respond to mechanical stimuli.

Drug screening in human physiologic medium identifies uric acid as an inhibitor of rigosertib efficacy

Rawat, V. — 2023-07-28

The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds, including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism waste product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. Structural modelling studies suggest that uric acid interacts with the tubulin-rigosertib complex and may act as an uncompetitive inhibitor of rigosertib. These results offer a possible explanation for the failure of rigosertib in clinical trials and demonstrate the utility of physiological media to achieve in vitro results that better represent human therapeutic responses.

Machine learning assisted health viability assay for mouse embryos with artificial confocal microscopy (ACM)

Goswami, N. — 2023-07-30

The combination of a good quality embryo and proper maternal health factors promise higher chances of a successful in vitro fertilization (IVF) procedure leading to clinical pregnancy and live birth. Of these two factors, selection of a good embryo is a controllable aspect. The current gold standard in clinical practice is visual assessment of an embryo based on its morphological appearance by trained embryologists. More recently, machine learning has been incorporated into embryo selection "packages". Here, we report a machine-learning assisted embryo health assessment tool utilizing a quantitative phase imaging technique called artificial confocal microscopy (ACM). We present a label-free nucleus detection method with novel quantitative embryo health biomarkers. Two viability assessment models are presented for grading embryos into two classes: healthy/intermediate (H/I) or sick (S) class. The models achieve a weighted F1 score of 1.0 and 0.99 respectively on the in-distribution test set of 72 fixed embryos and a weighted F1 score of 0.9 and 0.95 respectively on the out-of-distribution test dataset of 19 time-instances from 8 live embryos.

Modeling Allosteric Mechanisms of Eukaryotic Type II Topoisomerases

Evoli, S. — 2023-08-03

Type II topoisomerases (TopoIIs) are ubiquitous enzymes that are involved in crucial nuclear processes such as genome organization, chromosome segregation, and other DNA metabolic processes. These enzymes function as large, homodimeric complexes that undergo a complex cycle of binding and hydrolysis of two ATP molecules in their ATPase domains, which regulates the capture and passage of one DNA double-helix through a second, cleaved DNA molecule. This process requires the transmission of information about the state of the bound nucleotide over vast ranges in the TopoII complex. How this information is transmitted at the molecular level to regulate TopoII functions and how protein substitutions disrupt these mechanisms remains largely unknown. Here, we employed extensive microsecond scale molecular dynamics simulations of the yeast TopoII enzyme in multiple nucleotide-bound states and with amino acid substitutions near both the N- and C-terminals of the complex. Simulation results indicate that the ATPase domains are remarkably flexible on the sub-microsecond timescale and that these dynamics are modulated by the identity of the bound nucleotides and both local and distant amino acid substitutions. Network analyses point towards specific allosteric networks that transmit information about the hydrolysis cycle throughout the complex, which include residues in both the protein and the bound DNA molecule. Amino acid substitutions weaken many of these pathways. Together, our results provide molecular-level details on how the TopoII catalytic cycle is controlled through nucleotide binding and hydrolysis and how mutations may disrupt this process. SIGNIFICANCETypeII Topoisomerases (TopoIIs) are essential and ubiquitous for maintaining DNA topology in the nucleus. The mechanisms by which information about the nucleotide-binding state is transmitted from the ATPase domains throughout the TopoII complex remain poorly understood. We used microsecond timescale molecular dynamics simulations to probe the allosteric mechanisms underlying TopoII function. Results indicate remarkable flexibility of the ATPase domains on this timescale which is modulated by nucleotide binding and local and distant amino acid substitutions. Furthermore, we mapped the allosteric networks linking the ATPase and DNA cleavage domains, and connected them to the ATPase domain dynamics. Our findings provide molecular-level insights into how nucleotide binding and hydrolysis regulate the TopoII catalytic cycle and how mutations can disrupt these processes.

Tofacitinib effects profound seizure suppression through mimicking EZH2 antagonism of a pro-inflammatory gene network in epilepsy.

Hoffman, O. R. — 2023-08-08

All current drug treatments for epilepsy, a neurological disorder affecting over 50 million people(1, 2) merely treat symptoms, and a third of patients do not respond to medication. There are no disease modifying treatments that may be administered briefly to patients to enduringly eliminate spontaneous seizures and reverse cognitive deficits(3, 4). Applying network approaches to rodent models and human temporal lobectomy samples at both whole tissue and single-nuclei resolutions, we observe the well-characterized pattern of rapid induction and subsequent quenching exhibited of the JAK/STAT pathway within days of epileptogenic insult. This is followed by a resurgent activation weeks to months later with the onset of spontaneous seizures. Targeting the first wave of activation after epileptic insult does not prevent disease. However, brief inhibition of the second wave with CP690550 (Tofacitinib) (5, 6) enduringly suppresses seizures, rescues deficits in spatial memory, and alleviates epilepsy-associated histopathological alterations. Seizure suppression lasts for at least 2 months after the final dose. Using discovery-based transcriptomic analysis across models of epilepsy and validation of putative mechanisms with human data, we demonstrate a powerful approach to identifying disease modifying targets; this may be useful for other neurological disorders. With this approach, we find that reignition of inflammatory JAK/STAT3 signaling in chronic epilepsy opens a window for disease modification with the FDA-approved, orally available drug CP690550.

Re-education of myeloid immune cells to reduce regulatory T cell expansion and impede breast cancer progression

Gamage, H. E. V. — 2023-08-14

Immune checkpoint blockade (ICB) has revolutionized cancer therapy but has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. We demonstrate that NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the NLRP3 inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Loss of NR0B2 increased mammary tumor growth and metastasis. Small molecule agonists, including one developed here, reduced Treg expansion, reduced metastatic growth and improved the efficacy of ICB. This work identifies NR0B2 as a target to re-educate myeloid immune cells providing proof-of-principle that this cholesterol-homeostasis axis may have utility in enhancing ICB. Brief SummaryImmune therapy has been disappointing for breast cancer. NR0B2 within myeloid immune cells reduces the expansion of Tregs, a highly immune suppressive subtype historically challenging to target. NR0B2 within myeloid immune cells represses the inflammasome, leading to reduced Treg expansion and subsequent tumor growth/metastasis. Activation of NR0B2 with small molecule agonists, including one developed herein, attenuates tumor growth and metastasis in murine models of mammary cancer.

Molecular dynamics analyses of CLDN15 pore size and charge selectivity

McGuinness, S. — 2023-08-17

Claudin-15 (CLDN15) molecules form channels that directly regulate cation and water transport. In the gastrointestinal tract, this transport indirectly impacts nutrient absorption. However, the mechanisms governing ion transport through these channels remain poorly understood. We addressed this question by building on our previous cell culture studies and all atom molecular dynamic simulation model of CLDN15. By mutating D55 to a bulkier glutamic acid (E) or neutral amino acid asparagine (N), our in vitro measurements showed that the D55E mutation decreased charge selectivity and favored small ion permeability, while the D55N mutation led to reduced charge selectivity without markedly altering size selectivity. By establishing a simplified (reduced) CLDN15 molecular dynamics model that excludes non-essential transmembrane regions, we were able to probe how D55 modified cation dehydration, charge interaction, and permeability. These results provide novel insight into organization of the CLDN15 selectivity filter and suggests that D55 plays a dual role in shaping both electrostatic and steric properties of the pore, but its electrostatic role is more prominent in determining CLDN15 cation permeability. This knowledge can be used toward the development of effective strategies to modulate CLDN15 function. The experimental approach established can be further extended to study the function of other claudin channels. Together, these advancements will help us to modulate tight junctions to promote human health. SUMMARYCell culture and molecular dynamics simulations reveal the role of the CLDN15-D55 residue in ion size and charge selectivity. A reduced CLDN15 model offers novel insights into ion conductance, providing a valuable tool for therapeutic modulation of tight junctions.

Dual-specificity protein phosphatase 6 (DUSP6) overexpression reduces amyloid load and improves memory deficits in male 5xFAD mice

Pan, A. L. — 2023-08-25

BackgroundDual specificity protein phosphatase 6 (DUSP6) was recently identified as a key hub gene in a causal network that regulates late-onset Alzheimers disease. Importantly, decreased DUSP6 levels are correlated with an increased clinical dementia rating in human subjects, and DUSP6 levels are additionally decreased in the 5xFAD amyloidopathy mouse model. MethodsAAV5-DUSP6 or AAV5-GFP (control) were stereotactically injected into the dorsal hippocampus (dHc) of female and male 5xFAD or wild type mice to overexpress DUSP6 or GFP. Spatial learning memory of these mice was assessed in the Barnes maze, after which hippocampal tissues were isolated for downstream analysis. ResultsBarnes maze testing indicated that DUSP6 overexpression in the dHc of 5xFAD mice improved memory deficits and was associated with reduced amyloid plaque load, A{beta}1-40 and A{beta}1-42 levels, and amyloid precursor protein processing enzyme BACE1, in male but not in female mice. Microglial activation and microgliosis, which are increased in 5xFAD mice, were significantly reduced by dHc DUSP6 overexpression in both males and females. Transcriptomic profiling of female 5xFAD hippocampus revealed upregulated expression of genes involved in inflammatory and extracellular signal-regulated kinase (ERK) pathways, while dHc DUSP6 overexpression in female 5xFAD mice downregulated a subset of genes in these pathways. A limited number of differentially expressed genes (DEGs) (FDR<0.05) were identified in male mice; gene ontology analysis of DEGs (p<0.05) identified a greater number of synaptic pathways that were regulated by DUSP6 overexpression in male compared to female 5xFAD. Notably, the msh homeobox 3 gene, Msx3, previously shown to regulate microglial M1/M2 polarization and reduce neuroinflammation, was one of the most robustly upregulated genes in female and male wild type and 5xFAD mice overexpressing DUSP6. ConclusionsIn summary, our data indicate that DUSP6 overexpression in dHc reduced amyloid deposition and memory deficits in male but not female 5xFAD mice, whereas reduced neuroinflammation and microglial activation were observed in both males and females. The sex-dependent regulation of synaptic pathways by DUSP6 overexpression, however, correlated with the improvement of spatial memory deficits in male but not female 5xFAD.

SenePy: Unveiling the Cell-Type Specific Landscape of Cellular Senescence through Single-Cell Analysis in Living Organisms

Sanborn, M. A. — 2023-08-31

Senescent cells accumulate in tissues with organismal age and contribute causally to multiple chronic diseases. In vivo senescent cell phenotypes are heterogeneous because cellular context and stressors vary by cell type and tissue. Due to the variability of senescence programs, there is no universal method to identify senescent cells and even widely used markers, such as CDKN2A, are not ubiquitous. Therefore, we interrogated the Tabula Muris Senis mouse single-cell aging atlas and an array of single-cell datasets from human donors that spanned many ages to find cell-specific signatures of cellular senescence. We derived 75 mouse and 65 human senescence signatures from individual cell populations. CDKN2A and other markers of senescence were overrepresented in these signatures but there were many novel senescence genes present at higher rates. Within individual cell populations, we observed multiple programs of senescence with distinct temporal and transcriptional characteristics. We packaged the signatures along with a single-cell scoring method into an open-source package: SenePy. SenePy signatures better recapitulate cellular senescence than available methods when tested on multiple in vivo RNA-seq datasets and a p16ink4a reporter single-cell dataset. We used SenePy to map the kinetics of senescent cell accumulation across 97 cell types from humans and mice. SenePy also generalizes to disease-associate senescence and we used it to identify an increased burden of senescent cells in COVID-19 and myocardial infarction. This work provides a significant advancement towards our ability to identify and characterize in vivo cellular senescence.

Estrogen prevents age-dependent beige adipogenesis failure through NAMPT-controlled ER stress pathway

Park, J. — 2023-09-01

Thermogenic beige adipocytes are recognized as potential therapeutic targets for combating metabolic diseases. However, the metabolic advantages they offer are compromised with aging. Here, we show that treating mice with estrogen (E2), a hormone that decreases with age, to mice can counteract the aging- related decline in beige adipocyte formation when subjected to cold, while concurrently enhancing energy expenditure and improving glucose tolerance. Mechanistically, we find that nicotinamide phosphoribosyltranferase (NAMPT) plays a pivotal role in facilitating the formation of E2-induced beige adipocytes, which subsequently suppresses the onset of age-related ER stress. Furthermore, we found that targeting NAMPT signaling, either genetically or pharmacologically, can restore the formation of beige adipocytes by increasing the number of perivascular adipocyte progenitor cells. Conversely, the absence of NAMPT signaling prevents this process. In conclusion, our findings shed light on the mechanisms governing the age-dependent impairment of beige adipocyte formation and underscore the E2-NAMPT controlled ER stress as a key regulator of this process. HighlightsO_LIEstrogen restores beige adipocyte failure along with improved energy metabolism in old mice. C_LIO_LIEstrogen enhances the thermogenic gene program by mitigating age-induced ER stress. C_LIO_LIEstrogen enhances the beige adipogenesis derived from SMA+ APCs. C_LIO_LIInhibiting the NAMPT signaling pathway abolishes estrogen-promoted beige adipogenesis. C_LI

The RU486-dependent activation of the GeneSwitch system in adult muscles leads to severe adverse effects in Drosophila

Zappia, M. P. — 2023-09-07

Robust genetic systems to control the expression of transgenes in a spatial and temporal manner are a valuable asset for researchers. The GeneSwitch system induced by the drug RU486 has gained widespread use in the Drosophila community. However, some concerns were raised as negative effects were seen depending on the stock, transgene, stage and tissue under study. Here, we characterized the adverse effects triggered by activating the GeneSwitch system in adult muscles using the MHC-GS-GAL4 driver. When a control, mock UAS-RNAi transgene was induced by feeding adult flies with RU486, we found that the overall muscle structure, including myofibrils and mitochondrial shape, was significantly disrupted and led to a significant reduction in the lifespan. Remarkably, lifespan was even shorter when two copies of the driver were used even without the mock UAS-RNAi transgene. Thus, researchers should be cautious when interpreting the results given the adverse effects we found when inducing RU486-dependent MHC-GS-GAL4 in adult muscles. To counter the impact of these effects we recommend setting up additional control groups, such as a mock UAS-RNAi transgene, to validate the findings when using this inducible genetic system, as comparing the phenotypes between RU486-treated and untreated animals could be insufficient.

Maternal IL-10 restricts fetal emergency myelopoiesis

Collins, A. — 2023-09-13

Neonates, in contrast to adults, are highly susceptible to inflammation and infection. Here we investigate how late fetal liver (FL) mouse hematopoietic stem and progenitor cells (HSPC) respond to inflammation, testing the hypothesis that deficits in engagement of emergency myelopoiesis (EM) pathways limit neutrophil output and contribute to perinatal neutropenia. We show that despite similar molecular wiring as adults, fetal HSPCs have limited production of myeloid cells at steady state and fail to activate a classical EM transcriptional program. Moreover, we find that fetal HSPCs are capable of responding to EM-inducing inflammatory stimuli in vitro, but are restricted by maternal anti-inflammatory factors, primarily interleukin-10 (IL-10), from activating EM pathways in utero. Accordingly, we demonstrate that loss of maternal IL-10 restores EM activation in fetal HSPCs but at the cost of premature parturition. These results reveal the evolutionary trade-off inherent in maternal anti-inflammatory responses that maintain pregnancy but render the fetus unresponsive to EM activation signals and susceptible to infection. HIGHLIGHTSO_LIThe structure of the HSPC compartment is conserved from late fetal to adult life. C_LIO_LIFetal HSPCs have diminished steady-state myeloid cell production compared to adult. C_LIO_LIFetal HSPCs are restricted from engaging in emergency myelopoiesis by maternal IL-10. C_LIO_LIRestriction of emergency myelopoiesis may explain neutropenia in septic neonates. C_LI eTOC BLURBFetal hematopoietic stem and progenitor cells are restricted from activating emergency myelopoiesis pathways by maternal IL-10, resulting in inadequate myeloid cell production in response to inflammatory challenges and contributing to neonatal neutropenia.

Attenuated incubation of ethanol-induced conditioned taste aversion in a model of dependence

Ramirez, L. A. — 2023-09-14

RationalePreclinical studies report attenuated ethanol-induced conditioned taste aversion (CTA) following chronic ethanol exposure, suggesting that tolerance develops to the aversive properties of ethanol. However, these studies are confounded by pre-exposure to the unconditioned stimulus (US; ethanol), which is well known to hinder conditioning. ObjectivesThis study was designed to determine whether chronic ethanol exposure produces tolerance to the aversive properties of ethanol in the absence of a US pre-exposure confound. MethodsCTA was performed in adult male and female Long-Evans rats by pairing 0.1% ingested saccharin with an intraperitoneal injection of ethanol (1.5 or 2.0 g/kg) or saline. Rats were then rendered ethanol dependent using chronic intermittent ethanol (CIE) vapor exposure. Controls were exposed to room air (AIR). The effect of chronic ethanol on CTA expression and reconditioning were examined following vapor exposure. ResultsPrior to vapor exposure, both sexes developed CTA to a comparable degree with 2.0 g/kg producing greater CTA than 1.5 g/kg ethanol. Following vapor exposure, AIR controls exhibited an increase in CTA magnitude compared to pre-vapor levels. This effect was absent in CIE-exposed rats. These group differences were eliminated upon re-conditioning after vapor exposure. ConclusionsThese data suggest that chronic ethanol does not facilitate tolerance to the aversive properties of ethanol but rather, attenuates incubation of ethanol-induced CTA. Loss of CTA incubation suggests that CIE exposure disrupts circuits encoding aversion.

RGS2 is an innate immune checkpoint for TLR4 and Gαq-mediated IFNγ generation and lung injury

Joshi, J. C. — 2023-09-23

IFN{gamma}, a type II interferon secreted by immune cells, augments tissue responses to injury following pathogenic infections leading to lethal acute lung injury (ALI). Alveolar macrophages (AM) abundantly express Toll-like receptor-4 and represent the primary cell type of the innate immune system in the lungs. A fundamental question remains whether AM generation of IFNg leads to uncontrolled innate response and perpetuated lung injury. LPS induced a sustained increase in IFNg levels and unresolvable inflammatory lung injury in the mice lacking RGS2 but not in RGS2 null chimeric mice receiving WT bone marrow or receiving the RGS2 gene in AM. Thus, indicating RGS2 serves as a gatekeeper of IFNg levels in AM and thereby lungs innate immune response. RGS2 functioned by forming a complex with TLR4 shielding Gaq from inducing IFNg generation and AM inflammatory signaling. Thus, inhibition of Gaq blocked IFNg generation and subverted AM transcriptome from being inflammatory to reparative type in RGS2 null mice, resolving lung injury. HighlightsO_LIRGS2 levels are inversely correlated with IFN{gamma} in ARDS patients AM. C_LIO_LIRGS2 in alveolar macrophages regulate the inflammatory lung injury. C_LIO_LIDuring pathogenic insult RGS2 functioned by forming a complex with TLR4 shielding Gq from inducing IFN{gamma} generation and AM inflammatory signaling. C_LI eToc BlurbAuthors demonstrate an essential role of RGS2 in macrophages in airspace to promoting anti-inflammatory function of alveolar macrophages in lung injury. The authors provided new insight into the dynamic control of innate immune response by Gq and RGS2 axis to prevent ALI.

Structural basis of Cfr-mediated antimicrobial resistance and mechanisms for its evasion

Aleksandrova, E. V. — 2023-09-28

The ribosome is an essential drug target as many classes of clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent mechanisms of resistance to PTC-acting drugs is C8-methylation of the universally conserved adenine residue 2503 (A2503) of the 23S rRNA by the methyltransferase Cfr. Despite its clinical significance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. In this work, we developed a method to express a functionally-active Cfr-methyltransferase in the thermophilic bacterium Thermus thermophilus and report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-tRNAs. Our structures reveal that an allosteric rearrangement of nucleotide A2062 upon Cfr-methylation of A2503 is likely responsible for the inability of some PTC inhibitors to bind to the ribosome, providing additional insights into the Cfr resistance mechanism. Lastly, by determining the structures of the Cfr-methylated ribosome in complex with the antibiotics iboxamycin and tylosin, we provide the structural bases behind two distinct mechanisms of evading Cfr-mediated resistance.

Pathogenic Morphological Signatures of Perturbations in Mitochondrial-Related Genes Revealed by Pooled Imaging Assay

Kremitzki, C. L. — 2023-10-02

Mutations in mitochondrial-related genes underlie numerous neurodegenerative diseases, yet the significance of most variants remains uncertain concerning disease phenotypes. Several thousand genes have been shown to regulate mitochondria in eukaryotic cells, but which of these genes are necessary for proper mitochondrial dynamics? We investigated the degree of morphological disruptions in mitochondrial gene-silenced cells to understand the magnitude of genetic contribution to properly functioning mitochondria and to identify pathogenic variants. We analyzed 5,835 gRNAs in a high dimensional phenotypic dataset produced by the image-based pooled analysis platform Raft-Seq. Using the MFN2-mutant cell phenotype, we identified several genes, including TMEM11, TIMM8A, and three NADH Ubiquinone proteins, as crucial for normal mitochondrial morphology in human U2OS cells. Additionally, we found several missense and UTR variants within the genes SLC25A19 and ATAD3A as drivers of mitochondrial aggregation. By examining multiple features instead of a single readout, this analysis was powered to detect genes which had morphological signatures aligned with MFN2-mutant phenotypes. Reanalysis with anomaly detection revealed other critical genes, including APOOL, MCEE, NIT, PHB, and SLC16A7, which perturb mitochondrial network morphology in a manner divergent from MFN2. These studies offer insights into the molecular basis for mitochondrial dysfunction, setting the stage for new genomic diagnostics and therapeutic discovery.

Using a modular massively parallel reporter assay to discover context-specific regulatory grammars in type 2 diabetes

Tovar, A. — 2023-10-10

Most genome-wide association signals for complex disease reside in the noncoding genome, where defining function is nontrivial. MPRAs (massively parallel reporter assays) offer a scalable means to identify functional regulatory elements, but are typically conducted without regard to cell type, pairing cloned fragments with a generic housekeeping promoter. To explore the context-sensitivity of MPRAs, we screened enhancer activity across a panel of nearly 12,000 198-bp fragments spanning over 300 type 2 diabetes- and metabolic trait-associated regions in the 832/13 rat insulinoma beta cell line, a relevant model of pancreatic beta cells. We explored these fragments context sensitivity by comparing their activities when placed up- or downstream of a reporter gene, and in combination with either a synthetic housekeeping promoter (SCP1) or a more biologically relevant promoter corresponding to the human insulin (INS) gene. We identified clear effects of MPRA construct design on enhancer activity. Specifically, a subset of fragments (n = 702/11,656) displayed positional bias, evenly distributed across up- and downstream preference. Promoter choice also influenced MPRA activity (n = 698/11,656), mostly biased towards the cell-specific INS promoter (73.4%). To identify sequence features associated with promoter preference, we used Lasso regression with 562 genomic annotations and discovered that fragments with INS promoter-biased activity are enriched for HNF1 motifs. HNF1 family transcription factors are key regulators of glucose metabolism disrupted in maturity onset diabetes of the young (MODY), suggesting genetic convergence between rare coding variants that cause MODY and common T2D-associated regulatory regions. We designed a follow-up MPRA containing HNF1 motif-enriched fragments and observed several instances where deletion or mutation of HNF1 motifs disrupted the INS promoter-biased enhancer activity, specifically in the beta cell model but not in a skeletal muscle cell line, another diabetes-relevant cell type. Together, our study suggests that cell-specific regulatory activity is partially influenced by enhancer-promoter compatibility and indicates that careful attention should be paid when designing MPRA libraries to capture context-specific regulatory processes at disease-associated genetic signals.

ASCT2 is the primary serine transporter in cancer cells

Conger, K. O. — 2023-10-11

The non-essential amino acid serine is a critical nutrient for cancer cells due to its diverse biosynthetic functions. While some tumors can synthesize serine de novo, others are auxotrophic for serine and therefore reliant on the uptake of exogenous serine. Importantly, however, the transporter(s) that mediate serine uptake in cancer cells are not known. Here, we characterize the amino acid transporter ASCT2 (coded for by the gene SLC1A5) as the primary serine transporter in cancer cells. ASCT2 is well-known as a glutamine transporter in cancer, and our work demonstrates that serine and glutamine compete for uptake through ASCT2. We further show that ASCT2-mediated serine uptake is essential for purine nucleotide biosynthesis and that ER promotes serine uptake by directly activating SLC1A5 transcription. Together, our work defines an additional important role for ASCT2 as a serine transporter in cancer and evaluates ASCT2 as a potential therapeutic target in serine metabolism.

Clinically relevant humanized mouse models of metastatic prostate cancer to evaluate cancer therapies

Kostlan, R. J. — 2023-10-18

There is tremendous need for improved prostate cancer (PCa) models. The mouse prostate does not spontaneously form tumors and is anatomically and developmentally different from the human prostate. Engineered mouse models lack the heterogeneity of human cancer and rarely establish metastatic growth. Human xenografts represent an alternative but rely on an immunocompromised host. Accordingly, we generated PCa murine xenograft models with an intact human immune system (huNOG and huNOG-EXL mice) to test whether humanizing tumor-immune interactions would improve modeling of metastatic PCa and the impact of hormonal and immunotherapies. These mice maintain multiple human cell lineages, including functional human T-cells and myeloid cells. In 22Rv1 xenografts, subcutaneous tumor size was not significantly altered across conditions; however, metastasis to secondary sites differed in castrate huNOG vs background-matched immunocompromised mice treated with enzalutamide (enza). VCaP xenograft tumors showed decreases in growth with enza and anti-Programed-Death-1 treatments in huNOG mice, and no effect was seen with treatment in NOG mice. Enza responses in huNOG and NOG mice were distinct and associated with increased T-cells within tumors of enza treated huNOG mice, and increased T-cell activation. In huNOG-EXL mice, which support human myeloid development, there was a strong population of immunosuppressive regulatory T-cells and Myeloid-Derived-Suppressor-Cells (MDSCs), and enza treatment showed no difference in metastasis. Results illustrate, to our knowledge, the first model of human PCa that metastasizes to clinically relevant locations, has an intact human immune system, responds appropriately to standard-of-care hormonal therapies, and can model both an immunosuppressive and checkpoint-inhibition responsive immune microenvironment.

Caveolin-1 mediates neuroinflammation and cognitive impairment in SARS-CoV-2 infection

Trevino, T. N. — 2023-10-19

Leukocyte infiltration of the CNS can contribute to neuroinflammation and cognitive impairment. Brain endothelial cells regulate adhesion, activation, and diapedesis of T cells across the blood-brain barrier (BBB) in inflammatory diseases. The integral membrane protein Caveolin-1 (Cav-1) critically regulates BBB permeability, but its influence on T cell CNS infiltration in respiratory viral infections is unknown. In this study, we sought to determine the role of Cav-1 at the BBB in neuroinflammation in a COVID-19 mouse model. We used mice genetically deficient in Cav-1 to test the role of this protein in T cell infiltration and cognitive impairment. We found that SARS-CoV-2 infection upregulated brain endothelial Cav-1. Moreover, SARS-CoV-2 infection increased brain endothelial cell vascular cell adhesion molecule-1 (VCAM-1) and CD3+ T cell infiltration of the hippocampus, a region important for short term learning and memory. Concordantly, we observed learning and memory deficits. Importantly, genetic deficiency in Cav-1 attenuated brain endothelial VCAM-1 expression and T cell infiltration in the hippocampus of mice with SARS-CoV-2 infection. Moreover, Cav-1 KO mice were protected from the learning and memory deficits caused by SARS-CoV-2 infection. These results indicate the importance of BBB permeability in COVID-19 neuroinflammation and suggest potential therapeutic value of targeting Cav-1 to improve disease outcomes.

A Pipeline for a Primate Projectome: mapping every individual myelinated axon across the whole brain

Wildenberg, G. A. — 2023-10-24

1We developed a pipeline to detail the morphology and complete trajectory of every long distance projecting neuron in large volumes of the non-human primate brain - a projectome. A projectome would organize how the hundreds of brain regions in a primate brain communicate with each other and provide a comprehensive classification of long distance neurons. The reason such projectomes do not exist already is that there is no single imaging technique with the resolution and sampling depth capable of producing one in primates. Here we address that gap by combining new ways to cut brains with minimal tissue loss, nanoscale x-ray microscopy imaging that reveals every individual myelinated axon (MA) and the location, size, and morphology of every neuron, and algorithms for tracing individual myelinated axons for millimeters to centimeters, across tissue gaps.

Rebalancing the motor circuit restores movement in a Caenorhabditis elegans model for TDP-43-toxicity

Koopman, M. — 2023-10-25

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia are caused by the abnormal accumulation of TAR DNA-binding protein 43 (TDP-43) in the cytoplasm of neurons. How TDP-43 accumulation leads to disease symptoms is not well-characterized. Here, we use a C. elegans model for TDP-43-induced toxicity to identify the biological mechanisms that lead to disease-related phenotypes. By applying deep behavioral phenotyping, we established a phenotypic fingerprint of TDP-43 worms. This fingerprint was compared to that of 294 C. elegans mutants, in which genes were mutated that are important for nervous system and muscle functioning. By using a computational clustering approach, we found that the release of acetylcholine and GABA was the primary defect in TDP-43 worms. We then functionally dissected the neuromuscular circuit to show that GABA transmission was more severely diminished compared to acetylcholine. Whereas the loss of GABA transmission was caused by a profound loss of GABA synapses, acetylcholine neurons appeared to be functionally silenced. Enhancing functional output of repressed acetylcholine neurons at the level of G-protein coupled receptors or through optogenetic stimulation restored neurotransmission, but inefficiently rescued locomotion. Surprisingly, rebalancing the excitatory and inhibitory input by simultaneous stimulation of GABA and acetylcholine input into muscles not only synergized the effects of boosting individual neurotransmitter systems, but instantaneously improved movement. Our results suggest that interventions accounting for the altered connectome may be more efficient in restoring motor function than those solely focusing on diseased neuron populations.

ARPE-19 Retinal Pigment Epithelial Cells Cultured under Subconfluent Conditions Display Mesenchymal Cell State Characteristics that Correlate with Permissivity to HCMV Infection.

Golconda, P. — 2023-10-28

Human cytomegalovirus (HCMV) has a broad cellular tropism and epithelial cells are important physiological targets during infection. The retinal pigment epithelial cell line ARPE-19 has been used to model HCMV infection in epithelial cells for decades and remains a commonly used cell-type for studying viral entry, replication, and the cellular response to infection. We previously found that ARPE-19 cells, despite being derived from an epithelial cell explant, express extremely low levels of canonical epithelial proteins, such as E-cadherin and EpCAM. Here, we perform comparative studies of ARPE-19 and additional epithelial cell lines with strong epithelial characteristics. We find that ARPE-19 cells cultured under subconfluent conditions resemble mesenchymal fibroblasts, rather than epithelial cells; consistent with previous studies showing that ARPE-19 cultures require extended periods of high confluency culture to maintain epithelial characteristics. By reanalyzing public gene expression data and using machine-learning, we find evidence that ARPE-19 cultures maintained across many labs exhibit mesenchymal characteristics and that the majority of studies employing ARPE-19 use them in a mesenchymal state. Lastly, by performing experimental HCMV infections across mesenchymal and epithelial cell lines, we find that ARPE-19 cells behave like mesenchymal fibroblasts, producing logarithmic yields of cell-free infectious progeny, while cell lines with strong epithelial character exhibit an atypical infectious cycle and naturally restrict the production of cell-free progeny. Our work highlights important characteristics of the ARPE-19 cell line and suggests that sub-confluent ARPE-19 cells may not be optimal for modeling epithelial infection with HCMV or other human viruses. It also suggests that HCMV biosynthesis and/or spread may occur quite differently in epithelial cells compared to mesenchymal cells. These differences could contribute to viral persistence or pathogenesis in epithelial tissues.

Endothelial stiffening induced by CD36-mediated lipid uptake leads to endothelial barrier disruption and contributes to atherosclerotic lesions

Aguilar, V. — 2023-11-09

BackgroundTo determine the impact of endothelial stiffening induced by CD36-mediated lipid uptake in the disruption of aortic endothelial barrier and development of atherosclerosis in mouse models of obesity and hypercholesterolemia. Approach and ResultsEndothelial-specific inducible downregulation of CD36 results in abrogating the stiffening of aortic endothelium induced by a short-term (6-8 weeks) high-fat Western diet in intact freshly isolated mouse aortas of Cdh5.CreERT2CD36fl/fl mice, as assessed by atomic force microscopy. No effect was observed on the stiffness of aortic vascular wall assessed in the same groups of mice by echocardiography. Prevention of WD-induced endothelial stiffening by the downregulation of endothelial CD36 was associated with a protective effect against endothelial barrier disruption, assessed by morphological analysis of VE-cadherin junctions and penetration of Evans blue dye into the aortic wall. These protective effects were independent of the changes in the serum lipid profiles. Furthermore, endothelial specific downregulation of CD36 in hypercholesterolemic Cdh5.CreERT2CD36fl/flLDLR-/- mice also led to significant decrease in endothelial stiffening after 4-5 months of high fat diet and a significant decrease in the areas of atherosclerotic lesion. In both models, significant endothelial stiffening was observed specifically in male mice, while female mice exhibited less endothelial stiffening and less severe atherosclerosic phenotype, consistent with endothelial stiffening playing an important role in aortic vascular disease in a sex-dependent way. Mechanistically, we show in vitro that CD36-mdiated uptake of long chain saturated fatty acids, particularly palmitic acid, induces endothelial stiffening via activation of RhoA/ROCK pathway. Moreover, palmitic acid-induced endothelial stiffening critically depends on the expression of a RhoA inhibitory protein, Rho-GDI-1. ConclusionsWe conclude that stiffening of the aortic endothelium by CD36-mediated uptake of fatty acids contributes significantly to WD-induced vascular dysfunction and atherosclerosis. We further propose that fatty acids may activate RhoA by inducing its dissociation from Rho-GDI-1.

Nucleoporin93 (Nup93) Limits Yap Activity to Prevent Endothelial Cell Senescence

Nguyen, T. D. — 2023-11-14

Endothelial cells (ECs) form the innermost lining of the vasculature and serve a pivotal role in preventing age-related vascular disease. Endothelial health relies on the proper nucleocytoplasmic shuttling of transcription factors via nuclear pore complexes (NPCs). Emerging studies report NPC degradation with natural aging, suggesting impaired nucleocytoplasmic transport in age-related EC dysfunction. We herein identify nucleoporin93 (Nup93), a crucial structural NPC protein, as an indispensable player for vascular protection. Endothelial Nup93 protein levels are significantly reduced in the vasculature of aged mice, paralleling observations of Nup93 loss when using in vitro models of endothelial aging. Mechanistically, we find that loss of Nup93 impairs NPC transport, leading to the nuclear accumulation of Yap and downstream inflammation. Collectively, our findings indicate maintenance of endothelial Nup93 as a key determinant of EC health, where aging targets endothelial Nup93 levels to impair NPC function as a novel mechanism for EC senescence and vascular aging.

Conserved 5-methyluridine tRNA modification modulates ribosome translocation

Jones, J. D. — 2023-11-12

While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA in E. coli, Trm2 in S. cerevisiae) exhibit altered tRNA modifications patterns. Furthermore, m5U54 deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that, relative to wild-type cells, trm2{Delta} cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.

Evolution is All You Need in Promoter Design and Optimization

Ren, R. — 2023-11-18

Predicting the strength of promoters and guiding their directed evolution is a crucial task in synthetic biology. This approach significantly reduces the experimental costs in conventional promoter engineering. Previous studies employing machine learning or deep learning methods have shown some success in this task, but their outcomes were not satisfactory enough, primarily due to the neglect of evolutionary information. In this paper, we introduce the Chaos-Attention net for Promoter Evolution (CAPE) to address the limitations of existing methods. We comprehensively extract evolutionary information within promoters using chaos game representation and process the overall information with DenseNet and Transformer. Our model achieves state-of-the-art results on two kinds of distinct tasks. The incorporation of evolutionary information enhances the models accuracy, with transfer learning further extending its adaptability. Furthermore, experimental results confirm CAPEs efficacy in simulating in silico directed evolution of promoters, marking a significant advancement in predictive modeling for prokaryotic promoter strength. Our paper also presents a user-friendly website for the practical implementation of in silico directed evolution on promoters.

Investigating Phylogenetic Conflict using Homolog Trees reveals the Genetic Basis of Phenotypic Convergence in Carnivorous Sundews

Robertson, H. M. — 2023-11-18

Phenotypic convergence is found across the tree of life, and morphological similarities in distantly related species are often presumed to have evolved independently. However, clarifying the origins of traits has recently highlighted the complex nature of evolution, as apparent convergent features often share similar genetic foundations. Hence, the tree topology of genes that underlie such traits frequently conflicts with the overall history of species relationships. This conflict creates both a challenge for systematists and an exciting opportunity to investigate the rich, complex network of information that connects molecular trajectories with trait evolution. Here we present a novel conflict identification program named CAnDI (Conflict And Duplication Identifier), which enables the analysis of conflict in homologous gene trees rather than inferred orthologs. We demonstrate that the analysis of conflicts in homologous trees using CAnDI yields more comparisons than in ortholog trees in six datasets from across the eukaryotic tree of life. Using the carnivorous trap of Caryophyllales, a charismatic group of flowering plants, as a case study we demonstrate that analysing conflict on entire homolog trees can aid in inferring the genetic basis of trait evolution: by dissecting all gene relationships within homolog trees, we find genomic evidence that the molecular basis of the pleisiomorphic mucilaginous sticky trap was likely present in the ancestor of all carnivorous Caryophyllales. We also show that many genes whose evolutionary trajectories group species with similar trap devices code for proteins contributing to plant carnivory and identify a LATERAL ORGAN BOUNDARY DOMAIN transcription factor as a possible candidate for regulating sticky trap development.

Massively parallel mutant selection identifies genetic determinants of Pseudomonas aeruginosa colonization of Drosophila melanogaster

Miles, J. — 2023-11-21

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats and cause disease in a variety of hosts, including plants, invertebrates, and mammals. Understanding how this bacterium is able to occupy wide-ranging niches is important for deciphering its ecology. We used transposon sequencing (Tn-Seq, also known as INSeq) to identify genes in P. aeruginosa that contribute to fitness during colonization of Drosophila melanogaster. Our results reveal a suite of critical factors, including those that contribute to polysaccharide production, DNA repair, metabolism, and respiration. Comparison of candidate genes with fitness determinants discovered in previous studies of P. aeruginosa identified several genes required for colonization and virulence determinants that are conserved across hosts and tissues. This analysis provides evidence for both the conservation of function of several genes across systems, as well as host-specific functions. These findings, which represent the first use of transposon sequencing of a gut pathogen in Drosophila, demonstrate the power of Tn-Seq in the fly model system and advance existing knowledge of intestinal pathogenesis by D. melanogaster, revealing bacterial colonization determinants that contribute to a comprehensive portrait of P. aeruginosa lifestyles across habitats. ImportanceDrosophila melanogaster is a powerful model for understanding host-pathogen interactions. Research with this system has yielded notable insights into mechanisms of host immunity and defense, many of which emerged from analysis of bacterial mutants defective for well-characterized virulence factors. These foundational studies - and advances in high-throughput sequencing of transposon mutants - support unbiased screens of bacterial mutants in the fly. To investigate mechanisms of host-pathogen interplay and exploit the tractability of this model host, we used a high-throughput, genome-wide mutant analysis to find genes that enable a pathogen, P. aeruginosa, to colonize the fly. Our analysis reveals critical mediators of P. aeruginosa establishment in its host, some of which are required across fly and mouse systems. These findings demonstrate the utility of massively parallel mutant analysis and provide a platform for aligning the fly toolkit with comprehensive bacterial genomics.

The potassium channel subunit KV1.8 (Kcna10) is essential for the distinctive outwardly rectifying conductances of type I and II vestibular hair cells

Martin, H. R. — 2023-11-22

In amniotes, head motions and tilt are detected by two types of vestibular hair cells (HCs) with strikingly different morphology and physiology. Mature type I HCs express a large and very unusual potassium conductance, gK,L, which activates negative to resting potential, confers very negative resting potentials and low input resistances, and enhances an unusual non-quantal transmission from type I cells onto their calyceal afferent terminals. Following clues pointing to KV1.8 (KCNA10) in the Shaker K channel family as a candidate gK,L subunit, we compared whole-cell voltage-dependent currents from utricular hair cells of KV1.8-null mice and littermate controls. We found that KV1.8 is necessary not just for gK,L but also for fast-inactivating and delayed rectifier currents in type II HCs, which activate positive to resting potential. The distinct properties of the three KV1.8-dependent conductances may reflect different mixing with other KV subunits that are reported to be differentially expressed in type I and II HCs. In KV1.8-null HCs of both types, residual outwardly rectifying conductances include KV7 (KCNQ) channels. Current clamp records show that in both HC types, KV1.8-dependent conductances increase the speed and damping of voltage responses. Features that speed up vestibular receptor potentials and non-quantal afferent transmission may have helped stabilize locomotion as tetrapods moved from water to land. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/563853v3_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1409188org.highwire.dtl.DTLVardef@12aaa0org.highwire.dtl.DTLVardef@18000e0org.highwire.dtl.DTLVardef@b995c5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Identification and Characterization of a Novel Nematode Pan Allergen (NPA) from Wuchereria bancrofti and their Potential Role in Human Filarial Tropical Pulmonary Eosinophilia (TPE)

Katru, S. C. — 2023-12-04

Tropical pulmonary eosinophilia (TPE) is a chronic respiratory syndrome associated with Lymphatic Filariasis (LF), a tropical parasitic infection of the human, transmitted by mosquitoes. A larval form of LF, the microfilariae trapped in the lungs of TPE subjects have a major role in initiating the TPE syndrome. To date, there are no reports on the potential allergen that is responsible for generating parasite-specific IgE in TPE. In this project, we screened a cDNA expression library of the microfilarial stages of Wuchereria bancrofti with monoclonal IgE antibodies prepared from subjects with clinical filarial infections. Our studies identified a novel molecule that showed significant sequence similarity to an allergen. A blast analysis showed the presence of similar proteins in a number of nematodes parasites. Thus, we named the molecule as Nematode Pan Allergen (NPA). Subsequent functional analysis showed that NPA is a potent allergen that can cause release of histamine from mast cells, induce secretion of proinflammatory cytokines from alveolar macrophages and promote accumulation of eosinophils, all of which occur in TPE lungs. Therefore, we believe that NPA may have a significant role in the pathology of the TPE syndrome.

Pseudomonas aeruginosa kills Staphylococcus aureus in a polyphosphate-dependent manner

Shah, R. S. — 2023-12-06

Due to their frequent coexistence in many polymicrobial infections, including in patients with burn or chronic wounds or cystic fibrosis, recent studies have started to investigate the mechanistic details of the interaction between the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus. P. aeruginosa rapidly outcompetes S. aureus under in vitro co-cultivation conditions, which is mediated by several of P. aeruginosas virulence factors. Here, we report that polyphosphate (polyP), an efficient stress defense system and virulence factor in P. aeruginosa, plays a role for the pathogens ability to inhibit and kill S. aureus in a contact-independent manner. We show that P. aeruginosa cells characterized by low polyP level are less detrimental to S. aureus growth and survival while the gram-positive pathogen is significantly more compromised by the presence of P. aeruginosa cells that produce high level of polyP. We show that the polyP-dependent phenotype could be a direct effect by the biopolymer, as polyP is present in the spent media and causes significant damage to the S. aureus cell envelope. However, more likely is that polyPs effects are indirect through the regulation of one of P. aeruginosas virulence factors, pyocyanin. We show that pyocyanin production in P. aeruginosa occurs polyP-dependent and harms S. aureus through membrane damage and the generation of reactive oxygen species, resulting in increased expression of antioxidant enzymes. In summary, our study adds a new component to the list of biomolecules that the gram-negative pathogen P. aeruginosa generates to compete with S. aureus for resources. IMPORTANCEHow do interactions between microorganisms shape the course of polymicrobial infections? Previous studies have provided evidence that the two opportunistic pathogens P. aeruginosa and S. aureus generate molecules that modulate their interaction with potentially significant impact on disease outcomes. Our study identified the biopolymer polyP as a new effector molecule that impacts P. aeruginosas interaction with S. aureus. We show that P. aeruginosa kills S. aureus in a polyP-dependent manner, which occurs primarily through the polyP-dependent production of the P. aeruginosa virulence factor pyocyanin. Our findings add a new role for polyP to an already extensive list of functions. A more in-depth understanding of how polyP influences interspecies interactions is critical, as targeting polyP synthesis in bacteria such as P. aeruginosa may have a significant impact on other microorganisms and potentially result in dynamic changes in the microbial composition.

Mesenchymal Wnts are required for morphogenetic movements of calvarial osteoblasts during apical expansion

Polsani, N. — 2023-12-05

Apical expansion of calvarial osteoblast progenitors from the cranial mesenchyme (CM) above the eye is integral for calvarial growth and enclosure of the brain. The cellular behaviors and signals underlying the morphogenetic process of calvarial expansion are unknown. During apical expansion, we found that mouse calvarial primordia have consistent cellular proliferation, density, and survival with complex tissue scale deformations, raising the possibility that morphogenetic movements underlie expansion. Time lapse light sheet imaging of mouse embryos revealed that calvarial progenitors intercalate in 3D to converge supraorbital arch mesenchyme mediolaterally and extend it apically. In contrast, progenitors located further apically exhibited protrusive and crawling activity. CM cells express non-canonical Wnt/Planar Cell Polarity (PCP) core components and calvarial osteoblasts are bidirectionally polarized. We found non-canonical ligand, Wnt5a-/- mutants have less dynamic cell rearrangements, protrusive activity, and a flattened head shape. Loss of cranial mesenchyme-restricted Wntless (CM-Wls), a gene required for secretion of all Wnt ligands, led to diminished apical expansion of OSX+ calvarial osteoblasts in the frontal bone primordia in a non-cell autonomous manner without perturbing proliferation or survival. Calvarial osteoblast polarization, progressive cell elongation and enrichment for actin cytoskeleton protein along the baso-apical axis were dependent on CM-Wnts. Thus, CM-Wnts regulate cellular behaviors during calvarial morphogenesis and provide tissue level cues for efficient apical expansion of calvarial osteoblasts. These findings also offer potential insights into the etiologies of calvarial dysplasias.

PiNUI: A Dataset of Protein-Protein Interactions for Machine Learning

Dubourg-Felonneau, G. — 2023-12-13

We introduce a novel dataset named PiNUI: Protein Interactions with Nearly Uniform Imbalance. PiNUI is a dataset of Protein-Protein Interactions (PPI) specifically designed for Machine Learning (ML) applications that offer a higher degree of representativeness of real-world PPI tasks compared to existing ML-ready PPI datasets. We achieve such by increasing the data size and quality, and minimizing the sampling bias of negative interactions. We demonstrate that models trained on PiNUI almost always outperform those trained on conventional PPI datasets when evaluated on various general PPI tasks using external test sets. PiNUI is available here.

Dynamic protein assembly and architecture of the large solitary membraneless organelle during germline development in the wasp Nasonia vitripennis

Kharel, K. — 2023-12-13

Germ cells in different animals assemble characteristic membraneless organelles referred to as germ granules, which contain RNA and proteins required for germline development. Typically, the germ granules are small spherical or amorphous cytoplasmic granules and often, they assemble around membrane-bound organelles such as nuclei, mitochondria and endoplasmic reticulum. In particular, in egg chambers of the fruit fly Drosophila, nurse cells assemble perinuclear granules, referred to as nuage, along with multiple small germ granules formed at the posterior pole of the oocyte (polar granules). Nuage is assembled in a very similar way in the wasp Nasonia vitripennis, despite the long evolutionary distance from Drosophila. In contrast, Nasonia forms a very different single germ granule, called the oosome, at the posterior, which is about 40 times larger than a homologous Drosophila polar granule. Here, using molecular and super-resolution imaging approaches, we provide insights into protein assembly and architecture of the oosome during germline development. Interestingly, unlike the fly, the wasp utilizes alternatively spliced RNA-helicase Vasa isoforms during germline development and oosome formation. The isoforms differ by an unstructured region, containing repeats of phenylalanine and glycine, that is similar to functional domains characteristic of nucleoporins. In addition, while other conserved components of germ granules, such as Oskar, Aubergine and Tudor proteins are recruited to the oosome, these polypeptides show a distinct and specific localization within the oosome. Of particular note, Tudor protein forms a shell encapsulating the oosome, while small Oskar/Vasa/Aubergine granules occur inside the oosome core. Also, in surprising contrast to Drosophila egg chambers, we found that a subset of the wasp nurse cells located in anterior show dramatic DNA damage and assemble higher levels of nuage than their posterior counterparts. The characteristics of two distinct nurse cell populations suggest a mechanistic link between the higher amounts of nuage assembled in anterior nurse cells and their need to silence transposable elements in the presence of double-strand DNA breaks. Our results point to the high degree of plasticity in the assembly of membraneless organelles, which adapt to specific developmental needs of different organisms, and suggest that novel molecular features of conserved proteins result in the unique architecture of the oosome in the wasp.

Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot

Roca, J. — 2017-10-19

The functions of RNA pseudoknots (PKs), which are minimal tertiary structural motifs and an integral part of several ribozymes and ribonucleoprotein complexes, are determined by their structure, stability and dynamics. Therefore, it is important to elucidate the general principles governing their thermodynamics/folding mechanisms. Here, we combine experiments and simulations to examine the folding/unfolding pathways of the VPK pseudoknot, a variant of the Mouse Mammary Tumor Virus (MMTV) PK involved in ribosomal frameshifting. Fluorescent nucleotide analogs (2-aminopurine and pyrrolocytidine) placed at different stem/loop positions in the PK, and laser temperature-jump approaches serve as local probes allowing us to monitor the order of assembly of VPK with two helices with different intrinsic stabilities. The experiments and molecular simulations show that at 50 mM KCl the dominant folding pathway populates only the more stable partially folded hairpin. As the salt concentration is increased a parallel folding pathway emerges, involving the less stable hairpin structure as an alternate intermediate. Notably, the flux between the pathways is modulated by the ionic strength. The findings support the principle that the order of PK structure formation is determined by the relative stabilities of the hairpins, which can be altered by sequence variations or salt concentrations. Our study not only unambiguously demonstrates that PK folds by parallel pathways, but also establishes that quantitative description of RNA self-assembly requires a synergistic combination of experiments and simulations.nnSIGNIFICANCEThe assembly mechanism of RNA, vital to describing their functions, depends on both the sequence and the metal ion concentration. How the latter influences the folding trajectories remains an important unsolved problem. Here, we examine the folding pathways of an RNA pseudoknot (PK) with key functional roles in transcription and translation, using a combination of experiments and simulations. We demonstrate that the PK, consisting of two helices with differing stabilities, folds by parallel pathways. Surprisingly, the flux between them is modulated by monovalent salt concentration. Our work shows that the order of assembly of PKs is determined by the relative stability of the helices, implying that the folding landscape can be controlled by sequence and ion concentration.

The hummingbird and the hawk-moth:Species distribution, geographical partitioning, and macrocompetition across the United States

Halloway, A. — 2017-11-02

We introduce a new concept called macrocompetition - defined as the mutual suppression of diversity/species richness of competing clades - and investigate evidence for its existence. To this end, we analyzed the distribution of two convergent nectarivorous families, hawk-moths and hummingbirds, over the continental United States to determine whether there is geographic partitioning between the families and its potential causes. Using stepwise regression, we tested for latitudinal and longitudinal biases in the species richness of both taxa and the potential role of 10 environmental variables in their distribution pattern. Hawk-moth species richness increases with longitude (eastward-bias) while that of hummingbirds declines (westward-bias). Similar geographic patterns can be seen across Canada, Mexico and South America. Hawk-moth species richness is positively correlated with higher overall temperatures (especially summer minimums), atmospheric pressure, and summer precipitation; hummingbird species richness is negatively correlated with atmospheric pressure and positively correlated with winter daily maxima. The species richness patterns reflect each familys respective anatomical differences and support the concept of macrocompetition between the two taxa. Hawk-moth species richness was highest in states with low elevation, summer-time flowering, and warm summer nights; hummingbird species richness is highest in the southwest with higher elevation, greater cool season flowering and high daytime winter temperatures. Hawk-moths and hummingbirds as distinct evolutionary technologies exhibit niche overlap and geographical partitioning. These are two of three indicators suggested by Brown and Davidson for inter-taxonomic competition. We intend the patterns revealed here to inspire further exploration into competition and community structuring between hawk-moths and hummingbirds.

The Use of Biogeographical Patterns to Assess Key Adaptations in Two Families of Birds

Halloway, A. — 2017-11-04

Adaptations can be thought of as evolutionary technologies which allow an organism to exploit environments. Among convergent taxa, adaptations may be largely equivalent with the taxa operating in a similar set of environmental conditions, divergent with the taxa operating in different sets of environmental conditions, or superior with one taxon operating within an extended range of environmental conditions than the other. With this framework in mind, we sought to characterize the adaptations of two convergent nectarivorous bird families, the New World hummingbirds (Trochilidae) and Old World sunbirds (Nectariniidae), by comparing their biogeography. Looking at their elevational and latitudinal gradients, hummingbirds not only extend into but also maintain species richness in more extreme environments. We suspect that hummingbirds have a superior key adaptation that sunbirds lack, namely a musculoskeletal architecture that allows for hovering. Through biogeographic comparisons, we have been able to assess and understand adaptations as evolutionary technologies among two convergent bird families, a process that should work for most taxa.

Coupling MALDI-TOF mass spectrometry protein and specialized metabolite analyses to rapidly discriminate bacterial function

Clark, C. M. — 2017-11-08

For decades, researchers have lacked the ability to rapidly correlate microbial identity with bacterial metabolism. Since specialized metabolites are critical to bacterial function and survival in the environment, we designed a data acquisition and bioinformatics technique (IDBac) that utilizes in situ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze protein and specialized metabolite spectra of single bacterial colonies from agar plates. We demonstrated the power of our approach by discriminating between two Bacillus subtilis colonies in under 30 minutes, which differ by a single genomic mutation, solely on the basis of their differential ability to produce cyclic peptide antibiotics surfactin and plipastatin. Next, we employed our IDBac technique to detect subtle intra-species differences in the production of metal scavenging acyl-desferrioxamines in a group of eight freshwater Micromonospora isolates that share >99% sequence similarity in the 16S rRNA gene. Finally, we employed our method to simultaneously extract protein and specialized metabolite MS profiles from unidentified species of Lake Michigan sponge-associated bacteria cultivated on an agar plate. In just 3 hours, we created hierarchical protein MS groupings of 11 environmental isolates (10 MS replicates each, for a total of 110 samples) that accurately mirrored phylogenetic groupings. We further distinguished isolates within these groupings, which share nearly identical 16S rRNA gene sequence identity, based on inter- and intra-species differences in specialized metabolite production. To our knowledge, IDBac is the first attempt to couple in situ MS analyses of protein content and specialized metabolite production to allow the distinction of closely related bacterial colonies.nnSignificanceMass spectrometry is a powerful technique that has been used to identify bacteria via protein content, and to assess bacterial function in an environment via analysis of specialized metabolites. However, until now these analyses have operated independently, and this has resulted in the inability to rapidly connect bacterial phylogenetic identity with patterns of specialized metabolism. To bridge this gap, we designed a MALDI-TOF mass spectrometry data acquisition and bioinformatics pipeline (IDBac) to discriminate both intact protein and specialized metabolite spectra directly from bacterial cells grown on agar. To our knowledge, this is the first technique that organizes bacteria into highly similar phylogenetic groups and allows for comparison of metabolic differences of hundreds of isolates in just a few hours.

Mutations in RPL3L and MYZAP increase risk of atrial fibrillation

Thorolfsdottir, R. B. — 2017-11-21

We performed a meta-analysis of genome-wide association studies on atrial fibrillation (AF) among 14,710 cases and 373,897 controls from Iceland and 14,792 cases and 393,863 controls from the UK Biobank, focusing on low frequency coding and splice mutations, with follow-up in samples from Norway and the US. We observed associations with two missense (OR=1.19 for both) and one splice-donor mutation (OR=1.52) in RPL3L, encoding a ribosomal protein primarily expressed in skeletal muscle and heart. Analysis of 167 RNA samples from the right atrium revealed that the splice donor mutation in RPL3L results in exon skipping. AF is the first disease associated with RPL3L and RPL3L is the first ribosomal gene implicated in AF. This finding is consistent with tissue specialization of ribosomal function. We also found an association with a missense variant in MYZAP (OR=1.37), encoding a component of the intercalated discs of cardiomyocytes, the organelle harbouring most of the mutated proteins involved in arrhythmogenic right ventricular cardiomyopathy. Both discoveries emphasize the close relationship between the mechanical and electrical function of the heart.

Predicting E. coli concentrations using limited qPCR deployments at Chicago beaches

Lucius, N. — 2018-01-19

Culture-based methods to measure Escherichia coli (E. coli) are used by beach administrators to inform whether bacteria levels represent an elevated risk to swimmers. Since results take up to 12 hours, statistical models are used to forecast bacteria levels in lieu of test results; however they underestimate days with elevated fecal indicator bacteria levels. Quantitative polymerase chain reaction (qPCR) tests return results within 3 hours but are 2 to 5 times more expensive than culture-based methods. This paper presents a prediction model which uses limited deployments of qPCR tested sites with inter-beach correlation to predict when bacteria will exceed acceptable thresholds. The model can be used to inform management decisions on when to warn residents or close beaches due to exposure to the bacteria. Using data from Chicago collected between 2006 and 2016, the model proposed in this paper increased sensitivity from 3.4 percent to 11.2 percent-a 230 percent increase. We find that the correlation between beaches are substantial enough to provide higher levels of precision and sensitivity to predictive models. Thus, limited deployments of qPCR testing can be used to deliver better predictions for beach administrators at lower cost and less complexity.

PopPhy-CNN: A Phylogenetic Tree Embedded Architecture for Convolution Neural Networks for Metagenomic Data

Reiman, D. — 2018-01-31

MotivationAccurate prediction of the host phenotype from a metgenomic sample and identification of the associated bacterial markers are important in metagenomic studies. We introduce PopPhy-CNN, a novel convolutional neural networks (CNN) learning architecture that effectively exploits phylogentic structure in microbial taxa. PopPhy-CNN provides an input format of 2D matrix created by embedding the phylogenetic tree that is populated with the relative abundance of microbial taxa in a metagenomic sample. This conversion empowers CNNs to explore the spatial relationship of the taxonomic annotations on the tree and their quantitative characteristics in metagenomic data.nnResultsPopPhy-CNN is evaluated using three metagenomic datasets of moderate size. We show the superior performance of PopPhy-CNN compared to random forest, support vector machines, LASSO and a baseline 1D-CNN model constructed with relative abundance microbial feature vectors. In addition, we design a novel scheme of feature extraction from the learned CNN models and demonstrate the improved performance when the extracted features are used to train support vector machines.nnConclusionPopPhy-CNN is a novel deep learning framework for the prediction of host phenotype from metagenomic samples. PopPhy-CNN can efficiently train models and does not require excessive amount of data. PopPhy-CNN facilities not only retrieval of informative microbial taxa from the trained CNN models but also visualization of the taxa on the phynogenetic tree.nnContactyagndai@uic.edunnAvailabilitySource code is publicly available at https://github.com/derekreiman/PopPhy-CNNnnSupplementary informationSupplementary data are available at Bioinformatics online.

Experimental Zika Virus Infection in the Pregnant Common Marmoset Induces Spontaneous Fetal Loss and Neurodevelopmental Abnormalities

Seferovic, M. — 2018-02-04

During its most recent outbreak across the Americas, Zika virus (ZIKV) was surprisingly shown to cause fetal loss and congenital malformations in acutely and chronically infected pregnant women. However, understanding the underlying pathogenesis of ZIKV congenital disease has been hampered by a lack of relevant in vivo experimental models. Here we present a candidate New World monkey model of ZIKV infection in pregnant marmosets that faithfully recapitulates human disease. ZIKV inoculation at the human-equivalent of early gestation caused an asymptomatic seroconversion, induction of type I/II interferon-associated genes and proinflammatory cytokines, and persistent viremia and viruria. Spontaneous pregnancy loss was observed 16-18 days post-infection, with extensive active placental viral replication and fetal neurocellular disorganization similar to that seen in humans. These findings underscore the key role of the placenta as a conduit for fetal infection, and demonstrate the utility of marmosets as a highly relevant model for studying congenital ZIKV disease and pregnancy loss.

A structural mechano-chemical model for dynamic instability of microtubule

Stewman, S. — 2018-03-29

Microtubules are a major component of the cytoskeleton and vital to numerous cellular processes. The central dogma of microtubules is that all their functions are driven by dynamic instability; understanding its key phenomena (i.e. catastrophe, rescue, pause, differential behaviors at the plus and minus ends) distilled from a myriad of experiments under a consistent and unified scheme, however, has been unattainable. Here, we present a novel statistical-physics-based model uniquely constructed from conformational states deduced from existing tubulin structures, with transitions between them controlled by steric constraints and mechanical energy of the microtubule lattice. This mechano-chemical model allows, for the first time, all the key phenomena of dynamic instability to be coherently reproduced by the corresponding kinetic simulations. Long-puzzling phenomena, such as aging, small GTP-cap size, fast catastrophe upon dilution and temperature-induced ribbon-to-tube transition of GMPCPP-tubulins, robustly emerge and thus can be understood with confidence.

Liquid-Liquid Phase Separation of Patchy Particles Illuminates Diverse Effects of Regulatory Components on Protein Droplet Formation

Nguemaha, V. — 2018-04-03

Recently many cellular functions have been associated with membraneless organelles, or protein droplets, formed by liquid-liquid phase separation (LLPS). Proteins in these droplets often contain RNA-binding domains, but the effects of RNA on LLPS have been controversial. To gain better understanding on the roles of RNA, here we used Gibbs-ensemble simulations to determine phase diagrams of two-component patchy particles, as models for mixtures of proteins with RNA or other regulatory components. Protein-like particles have four patches, with attraction strength{varepsilon} PP; regulatory particles experience mutual steric repulsion but have two attractive patches toward proteins, with the strength{varepsilon} PR tunable. At low{varepsilon} PR, the regulator, due to steric repulsion, preferentially partitions in the dispersed phase, thereby displacing the protein into the droplet phase and promoting LLPS. At moderate{varepsilon} PR, the regulator starts to partition and displace the protein in the droplet phase, but only to weaken bonding networks and thereby suppress LLPS. At{varepsilon} PR >{varepsilon} PP, the enhanced bonding ability of the regulator initially promotes LLPS, but at higher amounts, the resulting displacement of the protein suppresses LLPS. These results illustrate how RNA can have disparate effects on LLPS, thus able to perform diverse functions in different organelles.

Taxa-driven functional shifts associated with stormflow in an urban stream microbial community

Chaudhary, A. — 2018-04-13

Urban streams are susceptible to stormwater and sewage inputs that can impact their ecological health and water quality. Microbial communities in streams play important functional roles and their composition and metabolic potential can help assess ecological state and water quality. Although these environments are highly heterogenous, little is known about the influence of isolated perturbations, such as those resulting from rain events on urban stream microbiota. Here, we examined the microbial community composition and diversity in an urban stream during dry and wet weather conditions with both 16S rRNA gene sequencing across multiple years and shotgun metagenomics to more deeply analyze a single stormflow event. Metagenomics was used to assess population-level dynamics as well as shifts in the microbial community taxonomic profile and functional potential before and after a substantial rainfall. Results demonstrated general trends present in the stream under stormflow vs. baseflow conditions across years and seasons and also highlighted the significant influence of increased effluent flow following rain in shifting the stream microbial community from abundant freshwater taxa to those more associated with urban/anthropogenic settings. Shifts in the taxonomic composition were also linked to changes in functional gene content, particularly for transmembrane transport and organic substance biosynthesis. We also observed an increase in relative abundance of genes encoding degradation of organic pollutants and antibiotic resistance after rain. Overall, this study provided evidence of stormflow impacts on an urban stream microbiome from an environmental and public health perspective.nnImportanceUrban streams in various parts of the world are facing increased anthropogenic pressure on their water quality, and stormflow events represent one such source of complex physical, chemical and biological perturbations. Microorganisms are important components of these streams from both ecological and public-health perspectives, and analyzing the effect of such perturbations on the stream microbial community can help improve current knowledge on the impact such chronic disturbances can have on these water resources. This study examines microbial community dynamics during rain-induced stormflow conditions in an urban stream of the Chicago Area Waterway System. Additionally, using shotgun metagenomics we identified significant shifts in the microbial community composition and functional gene content following a high rainfall event, with potential environment and public health implications. Previous work in this area has been limited to specific genes/organisms or has not assessed immediate stormflow impact.

Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks.

Clarke, R. — 2018-04-13

The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double strand break (DSB), Cas9 remains stably bound to it. Here we show persistent Cas9 binding blocks access to DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches one direction towards the Cas9-DSB complex. By exploiting these RNA polymerase-Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of PAM sequences and a simple method of improving selection of highly active sgRNA for genome editing.

Community origins and regional differences in plasmid-mediated fluoroquinolone resistant Enterobacteriaceae infections in children

Logan, L. K. — 2018-04-16

BackgroundFluoroquinolones (FQs) are uncommonly prescribed in children, yet pediatric multidrug-resistant (MDR)-Enterobacteriaceae (Ent) infections often reveal FQ resistance (FQR). We sought to define the molecular epidemiology of FQR and MDR-Ent in children.nnMethodsA case-control analysis of children with MDR-Ent infections at 3 Chicago hospitals was performed. Cases were children with third-generation-cephalosporin-resistant (3GCR) and/or carbapenem-resistant (CR)-Ent infections. PCR and DNA analysis assessed bla and plasmid-mediated FQR (PMFQR) genes. Controls were children with 3GC and carbapenem susceptible-Ent infections matched by age, source and hospital. We assessed clinical-epidemiologic predictors of PMFQR Ent infection.nnResultsOf 169 3GCR and/or CR Ent isolates from children (median age 4.8 years), 85 were FQR; 56 (66%) contained PMFQR genes. The predominant organism was E. coli and most common bla gene bla CTX-M-1 group. In FQR isolates, PMFQR gene mutations included aac61b-cr, oqxA/B, qepA, and qnrA/B/D/S in 83%, 15%, 13% and 11% of isolates, respectively. FQR E. coli was often associated with phylogroup B2, ST43/ST131. On multivariable analysis, PMFQR Ent infections occurred mostly in outpatients (OR 33.1) of non-black-white-Hispanic race (OR 6.5). Residents of Southwest Chicago were >5 times more likely to have PMFQR-Ent infections than those in the reference region, while residence in Central Chicago was associated with a 97% decreased risk. Other demographic, comorbidity, invasive-device, antibiotic use, or healthcare differences were not found.nnConclusionsThe strong association of infection with MDROs showing FQR with patient residence rather than with traditional risk factors suggests that the community environment is a major contributor to spread of these pathogens in children.

DDX41 recognizes RNA/DNA retroviral reverse transcripts and is critical for in vivo control of MLV infection

Stavrou, S. — 2018-05-02

Host recognition of viral nucleic acids generated during infection leads to the activation of innate immune responses essential for early control of virus. Retrovirus reverse transcription creates numerous potential ligands for cytosolic host sensors that recognize foreign nucleic acids, including single-stranded RNA (ssRNA), RNA/DNA hybrids and double stranded DNA (dsDNA). We and others recently showed that the sensors cyclic GMP-AMP synthase (cGAS), dead-box helicase 41 (DDX41) and members of the Aim2-like receptor (ALR) family participate in the recognition of retroviral reverse transcripts. However, why multiple sensors might be required and their relative importance in in vivo control of retroviral infection is not known. Here we show that DDX41 primarily senses the DNA/RNA hybrid generated at the first step of reverse transcription, while cGAS recognizes dsDNA generated at the next step. We also show that both DDX41 and cGAS are needed for the anti-retroviral innate immune response to MLV and HIV in primary mouse macrophages and dendritic cells (DC). Using mice with macrophage- or -specific knockout of the DDX41 gene, we show that DDX41 sensing in DCs but not macrophages was critical for controlling in vivo MLV infection. This suggests that DCs are essential in vivo targets for infection, as well as for initiating the antiviral response. Our work demonstrates that the innate immune response to retrovirus infection depends on multiple host nucleic acid sensors that recognize different reverse transcription intermediates.nnImportanceViruses are detected by many different host sensors of nucleic acid, which in turn trigger innate immune responses, such as type I IFN production, required to control infection. We show here that at least two sensors are needed to initiate a highly effective innate immune response to retroviruses - DDX41, which preferentially senses the RNA/DNA hybrid generated at the first step of retrovirus replication and cGAS, which recognizes double-stranded DNA generated at the 2nd step. Importantly, we demonstrate using mice lacking DDX41 or cGAS, that both sensors are needed for the full antiviral response needed to control in vivo MLV infection. These findings underscore the need for multiple host factors to counteract retroviral infection.

Quantitation of single action potential-evoked Ca2+ signals in CA1 pyramidal neuron presynaptic terminals

Church, E. — 2018-06-04

Presynaptic Ca2+ evokes exocytosis, endocytosis, and short-term synaptic plasticity. However, Ca2+ flux and interactions at presynaptic molecular targets are difficult to determine, because imaging has limited resolution. We measured single varicosity presynaptic Ca2+ using Ca2+ dyes as buffers, and constructed models of Ca2+ dispersal. Action potentials evoked Ca2+ transients (peak amplitude, 789{+/-}39 nM, within 2 ms of stimulation; decay times, 119{+/-}10 ms) with little variation when measured with low-affinity dye. Endogenous Ca2+ buffering capacities, action potential-evoked free [Ca2+]{inverted exclamation} and total amounts entering terminals were determined using high-affinity Ca2+ dyes to buffer Ca2+ transients. These data constrained Monte Carlo (MCell) simulations of Ca2+ entry, buffering, and removal. Data were well-fit with simulations of experimentally-determined Ca2+ fluxes, buffered by simulated Calbindin28K. Simulations were consistent with clustered Ca2+ entry followed within 2 ms by diffusion throughout the varicosity. Repetitive stimulation caused free varicosity Ca2+ to sum. However, simulated in nanometer domains, its removal by pumps and buffering was negligible, while diffusion rates were high. Thus, Ca2+ within tens of nanometers of entry, did not accumulate during sequential stimuli. A model of synaptotagmin1-Ca2+ binding indicates that even with 10 M free varicosity Ca2+, synaptogmin1 must be within tens of nanometers of channels to ensure occupation of all its Ca2+ binding sites. Repetitive stimulation, which evokes short-term synaptic enhancement, does not modify probabilities of Ca2+ fully occupying synaptotagmin1s C2 domains, suggesting that enhancement is not mediated by Ca2+-synaptotagmin1. We conclude that at spatio-temporal scale of fusion machines, Ca2+ necessary for their activation is diffusion dominated.

Ultralarge Modulation of Single Wall Carbon Nanotube Fluorescence Mediated by Neuromodulators Adsorbed on Arrays of Oligonucleotide Rings

Beyene, A. G. — 2018-06-20

Non-covalent interactions between single-stranded DNA (ssDNA) oligonucleotides and single wall carbon nanotubes (SWNTs) have provided a unique class of tunable chemistries for a variety of applications. However, mechanistic insight into both the photophysical and intermolecular phenomena underlying their utility is lacking, resulting in obligate heuristic approaches for producing ssDNA-SWNT based technologies. In this work, we present an ultrasensitive "turn-on" nanosensor for neuromodulators dopamine and norepinephrine with strong {Delta}F/F0 of up to 3500%, a signal appropriate for in vivo imaging, and uncover the photophysical principles and intermolecular interactions that govern the molecular recognition and fluorescence modulation of this nanosensor synthesized from the non-covalent conjugation of (GT)6 ssDNA strands on SWNTs. The fluorescence modulation of the ssDNA-SWNT conjugate is shown to exhibit remarkable sensitivity to the ssDNA sequence chemistry, length, and surface density, providing a wealth of parameters with which to tune nanosensor dynamic range and strength of fluorescence turn-on. We employ classical and quantum mechanical molecular dynamics simulations to rationalize our experimental findings. Calculations show that (GT)6 ssDNA form ordered loops around SWNT, inducing periodic surface potentials that modulate exciton recombination lifetimes. Further evidence is presented to elucidate how analyte binding modulates SWNT fluorescence. We discuss the implications of our findings for SWNT-based molecular sensing applications.

Structural basis for cholesterol transport-like activity of the Hedgehog receptor Patched

Zhang, Y. — 2018-06-20

Hedgehog protein signals mediate tissue patterning and maintenance via binding to and inactivation of their common receptor Patched, a twelve-transmembrane protein that otherwise would suppress activity of the seven-transmembrane protein, Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. The extracellular domain mediates association of Patched monomers in an unusual dimeric architecture that implies curvature in the associated membrane. A central conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Patched expression indeed reduces cholesterol activity in the inner leaflet of the plasma membrane, in a manner antagonized by Hedgehog stimulation and with implications for regulation of Smoothened.

The Association between Cigarette Affordability and Consumption

HE, Y. — 2018-07-03

ObjectivesTo calculate cigarette affordability for 78 countries worldwide from 2001 to 2014 using the Relative Income Price (RIP) ratio defined as the percentage of per capita GDP required to purchase 100 packs of cigarettes using the lowest price from Economist Intelligence Unit (EIU) database, examine the association between cigarette affordability and cigarette consumption, and calculate the affordability elasticity of demand.nnDesign and MethodsRIP (2001-2014) was calculated for 16 low-income economies, 19 lower middle-income economies, 13 upper middle-income economies, and 30 high-income economies. Ordinary least square regressions were used to analyze the association between cigarette affordability and consumption.nnResults and ConclusionsPer capita consumption continued to rise in low-income countries and decreased slightly in lower middle-income countries as the RIP of cigarette consistently declined in low- and lower middle-income economies from 2001 to 2014. The real cigarette prices continued to decline in low- and lower middle-income countries and continued to rise in upper middle- and high-income countries. Though cigarettes were more expensive in HICs than were in LMICs, cigarettes were more affordable in HICs than were in LMICs. The regression results show a 10% increase in the RIP of cigarettes led to a 2% decrease in per capita consumption. The affordability elasticity of demand differed significantly between HICs and LMICs. However, the effect of cigarette affordability on consumption has not changed over time. To control the smoking epidemic, low- and lower middle-income countries should further increase cigarette prices. The rate of price increase should exceed the rate of economic growth and outpace the inflation rate to make cigarettes less affordable and thereby reducing tobacco use.

Divergent Behavior Amid Convergent Evolution: A Case of Four Desert Rodents Learning to Respond to Known and Novel Vipers

Bleicher, S. S. — 2018-07-04

Desert communities word-wide are used as natural laboratories for the study of convergent evolution, yet inferences drawn from such studies are necessarily indirect. Here, we brought desert organisms together (rodents and vipers) from two deserts (Mojave and Negev). Both predators and prey in the Mojave have adaptations that give them competitive advantage compared to their middle-eastern counterparts. Heteromyid rodents, kangaroo rats and pocket mice, have fur-lined cheek pouches that allow the rodents to carry larger loads under predation risk compared to gerbilline rodents. Sidewinder rattlesnakes have heat-sensing pits, allowing them to hunt better on moonless nights when their Negev sidewinding counterpart, the Saharan horned vipers, are visually impaired. In behavioral-assays, we used giving-up density (GUD) to gage how each species of rodent perceived risk posed by known and novel snakes. We repeated this for the same set of rodents at first encounter and again two months later following intensive "natural" exposure to both snake species. Pre-exposure, all rodents identified their evolutionarily familiar snake as a greater risk than the novel one. However, post-exposure all identified the heat-sensing sidewinder rattlesnake as a greater risk. The heteromyids were more likely to avoid encounters with, and discern the behavioral difference among, snakes than their gerbilline counterparts.

Evaluating bacterial and functional diversity of human gut microbiota by complementary metagenomics and metatranscriptomics

Ranjan, R. — 2018-07-06

It is well accepted that dysbiosis of microbiota is associated with disease; however, the biological mechanisms that promote susceptibility or resilience to disease remain elusive. One of the major limitations of previous microbiome studies has been the lack of complementary metatranscriptomic (functional) data to complement the interpretation of metagenomics (bacterial abundance). The purpose of the study was twofold, first to evaluate the bacterial diversity and differential gene expression of gut microbiota using complementary shotgun metagenomics (MG) and metatranscriptomics (MT) from same fecal sample. Second, to compare sequence data using different Illumina platforms and with different sequencing parameters as new sequencers are introduced and determine if the data are comparable on different platforms. In this study, we perform ultra-deep metatranscriptomic shotgun sequencing for a sample that we previously analyzed with metagenomics shotgun sequencing. We validated the sequencing and analysis methods using different Illumina platform, and with different sequencing and analysis parameters. Our results suggest that use of different Illumina platform did not lead to detectable bias in the sequencing data. The analysis of the sample using MG and MT approach shows that some species genes are more highly represented in the MT than in the MG, indicating that some species are highly metabolically active. Our analysis also shows that ~52% of the genes in the metagenome are in the metatranscriptome, and therefore are robustly expressed. The functions of the low and rare abundance bacterial species remain poorly understood. Our observations indicate that among the low abundant species analyzed in this study some were found to be more metabolically active compared to others and can contribute distinct profiles of biological functions that may modulate the host-microbiota and bacteria-bacteria interactions.

Host Enzymes Heparanase and Cathepsin L Promote Herpes Simplex Virus-2 Release from Cells

Hopkins, J. — 2018-07-08

Herpes simplex virus-2 (HSV-2) can productively infect many different cell types of human and non-human origin. Here we demonstrate interconnected roles for two host enzymes, heparanase (HPSE) and cathepsin L in HSV-2 release from cells. In vaginal epithelial cells and other cell lines tested, HSV-2 causes heparan sulfate shedding and upregulation in HPSE levels during the productive phase of infection. We also noted increased levels of cathepsin L and show that regulation of HPSE by cathepsin L via cleavage of HPSE proenzyme is important for infection. Furthermore, inhibition of HPSE by a specific inhibitor, OGT 2115, dramatically reduces HSV-2 release from vaginal epithelial cells. Likewise, we show evidence that the inhibition of cathepsin L is detrimental to the infection. The HPSE increase after infection is mediated by an increased NF-{kappa}B nuclear localization and a resultant activation of HPSE transcription. Together these mechanisms contribute to the removal of heparan sulfate from the cell surface, and thus facilitate virus release from cells.nnImportanceGenital infections by HSV-2 represent one of the most common sexually transmitted viral infections. The virus causes painful lesions, and sores around the genitals or rectum. Intermittent release of the virus from infected tissues during sexual activities is the most common cause of transmission. At the molecular level, cell surface heparan sulfate (HS) is known to provide attachment sites for HSV-2. While the removal of HS during HSV-1 release has been shown, not much is known about the host factors and their regulators that contribute to HSV-2 release from natural target cell types. Here we suggest a role for the host enzyme heparanase in HSV-2 release. Our work reveals that in addition to the regulation of transcription by NF-{kappa}B, HPSE is also regulated post-translationally by cathepsin L and that inhibition of heparanase activity directly affects HSV-2 release. We provide unique insights into the host mechanisms controlling HSV-2 egress and spread.

Transcriptional Profiling of Patient Isolates Identifies a Novel TOR Regulatory Pathway in Cryptococcal Virulence

Park, Y.-D. — 2018-07-13

Human infection with Cryptococcus causes up to a quarter million AIDS-related deaths annually and is the most common cause of non-viral meningitis in the United States. As an opportunistic fungal pathogen, C. neoformans is distinguished by its ability to adapt to diverse host environments including plants, amoeba and mammals. In the present study, comparative transcriptomics of the fungus within human cerebrospinal fluid identified expression profiles representative of low-nutrient adaptive responses. Transcriptomics of fungal isolates from a cohort of HIV/AIDS patients identified a low nutrient-induced gene, an alternative carbon nutrient transporter STL1 associated with poor early fungicidal activity, an important clinical prognostic marker. Mouse modeling and pathway analysis demonstrated a role for STL1 in mammalian pathogenesis and revealed that STL1 expression is regulated by a novel target-of-rapamycin (TOR)-related multi-gene regulatory mechanism involving the CAC2 subunit of the chromatin assembly complex 1, CAF-1. In this pathway, the TOR-related RNA chaperone, VAD1 was found to transcriptionally regulate a cryptococcal homolog of a cytosolic protein Ecm15, in turn, required for nuclear transport of the Cac2 protein. Derepression of STL1 by the CAC2-containing CAF-1 complex was mediated by Cac2 and modulated binding and suppression of the STL1 enhancer element. Derepression of STL1 resulted in enhanced survival and growth of the fungus in the presence of low nutrient, alternative carbon sources, facilitating virulence in mice. The study underscores the utility of ex vivo expression profiling of fungal clinical isolates and provides fundamental genetic understanding of saprophyte adaption to the human host.nnAuthor summaryThe fungus Cryptococcus is a fungal pathogen that kills an estimated quarter of a million individuals yearly and is the most common cause of meningitis in the United States. The fungus is carried in about 10% of the adult population and, after re-activation, causes disease in a wide variety of individuals including HIV-infected as well as immunosuppression either from genetic defects or after immune suppressive treatments due to transplant conditioning, cancer therapy or treatment of autoimmune diseases. The fungus is widely carried in the soil and trees and can infect plants, single cell organisms and even dolphins. However, mechanisms for this widespread ability to infect a variety of hosts are poorly understood. The present study identified adaptation to low nutrients as a key property that allows the fungus to infect these diverse hosts and identified a nutrient transporter, STL1 to be associated with a marker of poor clinical outcome in a cohort of HIV/AIDS patients. Understanding molecular mechanisms involved in environmental adaptation may help to design better methods of control and treatment of widely dispersed fungal pathogens such as Cryptococcus.

Protein Structural Biology Using Cell-Free Platform from Wheat Germ

Novikova, I. V. — 2018-07-23

One of the biggest bottlenecks for structural analysis of proteins remains the creation of high yield and high purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multistage eukaryotic wheat-germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also developed a robust purification procedure, which can produce highly-pure (>98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid generation of samples for structural characterization that may not be amenable to other recombinant approaches.

Mechanical Behavior Of Axonal Microtubules; The Effect Of Fluid On The Rupture Of Axonal Microtubules

Manuchehrfar, F. — 2018-07-30

Axonal microtubules are dynamically instable bundles in the interior part of the axon. The dynamics of these bundles are of vital importance in the behavior of axon such as their degeneration. Each axon typically contains 10~100 microtubule bundles with average length of 4m. These bundles are coated with cytoplasm and are cross linked with random number of tau proteins. In some circumstances such as acceleration or deceleration of head in space or during the strike, they are placed in tension which may cause rupture of these bundles or disconnection of tau protein cross links. Mechanical behavior and rupture modality of microtubule bundles are becoming more and more important recently. In our model, viscoelastic microtubule bundles constituted from several discrete masses connected to the neighboring mass with a standard linear solid (SLS), a spring damper model. In addition we take into account the effect of cytoplasm by Dissipative Particle Dynamic (DPD) to investigate the rupture nature and mechanical behavior of these bundles and the effect of cytoplasm on their mechanical behavior. We obtain these results for various amounts of suddenly applied end forces to the group of axonal microtubule bundles.

Considerations for imaging thick, low contrast, and beam sensitive samples with liquid cell transmission electron microscopy

Moser, T. H. — 2018-07-30

Transmission electron microscopy of whole cells is hindered by the inherently large thickness and low atomic contrast intrinsic of cellular material. Liquid cell transmission electron microscopy allows samples to remain in their native hydrated state and may permit visualizing cellular dynamics in-situ. However, imaging biological cells with this approach remains challenging and identifying an optimal imaging regime using empirical data would help foster new advancements in the field. Recent questions about the role of the electron beam inducing morphological changes or damaging cellular structure and function necessitates further investigation of electron beam-cell interactions, but is complicated by variability in imaging techniques used across various studies currently present in literature. The necessity for using low electron fluxes for imaging biological samples requires finding an imaging strategy which produces the strongest contrast and signal to noise ratio for the electron flux used. Here, we experimentally measure and evaluate signal to noise ratios and damage mechanisms between liquid and cryogenic samples for cells using multiple electron imaging modalities all on the same instrument and with equivalent beam parameters to standardize the comparison. We also discuss considerations for optimal electron microscopy imaging conditions for future studies on whole cells within liquid environments.

Static kinks or flexible hinges: conformational distributions of bent DNA bound to integration host factor mapped by fluorescence lifetime measurements

Connolly, M. — 2018-08-02

Gene regulation depends on proteins that bind to specific DNA sites. Such specific recognition often involves severe DNA deformations including sharp kinks. It has been unclear how rigid or flexible these protein-induced kinks are. Here, we investigated the dynamic nature of DNA in complex with integration host factor (IHF), a nucleoid-associated architectural protein known to bend one of its cognate sites (35 base pair H) into a U-turn by kinking DNA at two sites. We utilized fluorescence lifetime based FRET spectroscopy to map the distribution of bent conformations in various IHF-DNA complexes. Our results reveal a surprisingly dynamic specific complex: while 80% of the IHF-H population exhibited FRET efficiency consistent with the crystal structure, 20% exhibited FRET efficiency indicative of unbent or partially bent DNA. This conformational flexibility is modulated by sequence variations in the cognate site. In another site (H1) that lacks an A-tract of H on one side of the binding site, the population in the fully U-bent conformation decreased to 36%, as did the extent of bending. A similar decrease in the U-bent population was observed with a single base mutation in H in a consensus region on the other side. Taken together, these results provide important insights into the finely tuned interactions between IHF and its cognate sites that keep the DNA bent (or not), and yield quantitative data on the dynamic equilibrium between different DNA conformations (kinked or not kinked) that depend sensitively on DNA sequence and deformability. Notably, the difference in dynamics between IHF-H and IHF-H1 reflects the different roles of these complexes in their natural context, in the phage lambda "intasome" (the complex that integrates phage lambda into the E. coli chromosome).

Opposing Roles of the Fork-head box genes FoxM1 and FoxA2 in Hepatocellular Carcinoma

Raychaudhuri, P. — 2018-08-02

The fork-head box transcription factor FoxMl is essential for hepatocellular carcinoma (HCC) development and its overexpression coincides with poor prognosis. Here, we show that the mechanisms by which FoxM1 drives HCC progression involve overcoming the inhibitory effects of the liver differentiation gene FoxA2. First, the expression patterns of FoxM1 and FoxA2 in human HCC are opposite. We show that FoxM1 represses expression of FoxA2 in G1 phase, a phase in the cell cycle in which cells can undergo differentiation. Repression of FoxA2 in G1 phase is important, as it is capable of inhibiting expression of the pluripotency genes that are expressed mainly in S/G2 phases. Using a transgenic mouse model for oncogenic Ras-driven HCC, we provide genetic evidence for a repression of FoxA2 by FoxM1. Conversely, FoxA2 inhibits expression of FoxM1, and inhibits FoxM1-induced tumorigenicity of HCC cells. Moreover, expression of FoxA2 in mouse liver expressing activated Ras inhibits FoxM1 expression and inhibits HCC progression. The observations provide strong genetic evidence for an opposing role of FoxM1 and FoxA2 in HCC progression.nnAUTHOR SUMMARYLiver cancer remains untreatable because it is diagnosed at a stage when the cancer is aggressive and resistant to therapeutics. The mechanism that drives aggressive liver cancer is poorly understood. These cancers are made up of poorly differentiated cancer cells. Interestingly, the FoxM1 gene is overexpressed in the aggressive liver cancers. Although FoxM1 is important for expression of the proliferation genes, it does not explain why it is overexpressed mainly in the undifferentiated cancers. The current study addresses this puzzle. Our previous studies demonstrated that FoxM1 increases expression of the pluripotency genes that are expressed mainly in the stem-like cells. In the current manuscript we show that, in addition to activating the pluripotency genes, FoxM1 inhibits expression of the liver differentiation gene FoxA2. Overexpression of FoxM1 is important for this inhibition function, as it involves the retinoblastoma family of proteins, which are often inactivated in cancer cells, and thus, are of low-abundance. Moreover, the inhibition of FoxA2 is significant because FoxA2 could inhibit expression of the pluripotency genes as well as FoxM1. The observations provide new insights into how FoxM1 drives progression of aggressive liver cancer.

Ankyrin domain encoding genes resulting from an ancient horizontal transfer are functionally integrated into developmental gene regulatory networks in the wasp Nasonia

Lynch, J. — 2018-08-02

BackgroundHow and why regulatory networks incorporate additional components, and how novel genes are maintained and functionally integrated into developmental processes are two important and intertwined questions whose answers have major implications for the evolution of development. We recently described a set of novel genes with robust and unique expression patterns along the dorsal-ventral axis of the embryo of the wasp Nasonia. Given the unique evolutionary history of these genes, and their apparent integration in to the dorsal-ventral (DV) patterning network, they are collectively an excellent model to study the evolution of regulatory networks, and the fates of novel genes.nnResultsWe have found that the novel DV genes are part of a large family of rapidly duplicating and diverging ankyrin domain encoding genes that originated most likely by horizontal transfer from Wolbachia in a common ancestor of the wasp superfamilly Chalcidoidea. We tested the function of those ankyrin encoding genes expressed along the DV axis and found that they participate in early embryonic DV patterning. We also developed a new wasp model system (Melittobia) and found that some functional integration of ankyrin genes have been preserved for over 90 million years, while others are lineage specific.nnConclusionsOur results indicate that regulatory networks can incorporate novel genes that then become necessary for stable and repeatable outputs. Even modest role in developmental networks may be enough to allow novel or duplicate genes to be maintained in the genome and become fully integrated network components.

Transcriptomic and functional analysis of the oosome, a unique form of germplasm in the wasp Nasonia vitripennis

Lynch, J. A. — 2018-08-03

BackgroundThe oosome is the germline determinant in the wasp Nasonia vitripennis and is homologous to the polar granules of Drosophila. Despite a common evolutionary origin and developmental role, the oosome is morphologically quite distinct from polar granules. It is a solid sphere that migrates within the cytoplasm before budding out and forming pole cells.nnResultsTo gain an understanding of both the molecular basis of the novel form of the oosome, and the conserved essential features of germ plasm, we quantified and compared transcript levels between embryo fragments that contained the oosome, and those that did not. The identity of the localized transcripts indicated that Nasonia uses different molecules to carry out conserved germ plasm functions. In addition, functional testing of a sample of localized transcripts revealed potentially novel mechanisms of ribonucleoprotein assembly and pole cell cellularization in the wasp.nnConclusionsOur results demonstrate that numerous novel and unexpected molecules have been recruited in order produce the unique characteristics of the oosome and pole cell formation in Nasonia. This work will serve as the basis for further investigation into the patterns of germline determinant evolution among insects, the molecular basis of extreme morphology of ribonucleoproteins, and the incorporation of novel components into developmental networks.

Assessing placental maturity through histological and transcriptomic analyses in idiopathic spontaneous preterm birth

Brockway, H. M. — 2018-08-07

Preterm birth (PTB) is leading contributor to infant death in the United States and globally, yet the underlying mechanistic causes are not well understood. Histopathological studies of preterm birth suggest advanced villous maturity may have a role in idiopathic spontaneous preterm birth (isPTB). To better understand pathological and molecular basis of isPTB, we compared placental villous transcriptomes from carefully phenotyped cohorts of PTB due to infection or isPTB between 28-36 weeks gestation and healthy term placentasu. Transcriptomic analyses revealed a unique expression signature for isPTB distinct from the age-matched controls that were delivered prematurely due to infection. This signature included the upregulation of three IGF binding proteins (IGFBP1, IGFBP2, and IGFBP6), supporting a role for aberrant IGF signaling in isPTB. However, within the isPTB expression signature, we detected secondary signature of inflammatory markers including TNC, C3, CFH, and C1R, which have been associated with placental maturity. In contrast, the expression signature of the gestational age-matched infected samples included upregulation of proliferative genes along with cell cycling and mitosis pathways. Together, these data suggest an isPTB molecular signature of placental hypermaturity, likely contributing to the premature activation of inflammatory pathways associated with birth and providing a molecular basis for idiopathic spontaneous birth.

NMDA Receptor Dysregulation by Defective Depalmitoylation in the Infantile Neuronal Ceroid Lipofuscinosis Mouse Model

Koster, K. P. — 2018-08-13

Protein palmitoylation and depalmitoylation alter protein function. This post-translational modification is critical for synaptic transmission and plasticity. Mutation of the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (PPT1) causes infantile neuronal ceroid lipofuscinosis (CLN1), a pediatric neurodegenerative disease. However, the role of protein depalmitoylation in synaptic maturation is unknown. Therefore, we studied synapse development in Ppt1-/- mouse visual cortex. We demonstrate the stagnation of the developmental N-methyl-D-aspartate receptor (NMDAR) subunit switch from GluN2B to GluN2A in Ppt1-/- mice. Correspondingly, GluN2A-mediated synaptic currents are diminished and Ppt1-/- dendritic spines maintain immature morphology in vivo. Further, GluN2B is hyperpalmitoylated in Ppt1-/- neurons and associated with extrasynaptic, diffuse calcium influxes and enhanced vulnerability to NMDA-induced excitotoxicity. Remarkably, Ppt1-/- neurons treated with palmitoylation inhibitors demonstrate normalized levels of palmitoylated GluN2B and Fyn kinase, reversing susceptibility to excitotoxic insult. Thus, depalmitoylation of GluN2B by PPT1 plays a critical role in postsynapse maturation and pathophysiology of neurodegenerative disease.

Gut microbial features can predict host phenotype response to protein deficiency

Navarro, G. — 2018-08-22

Malnutrition remains a major health problem in low and middle income countries. During low protein intake, < 0.67 g/kg/day, there is a loss of nitrogen (N2) balance, due to the unavailability of amino acid for metabolism and unbalanced protein catabolism results. However, there are individuals, who consume the same low protein intake, and preserve N2 balance for unknown reasons. A novel factor, the gut microbiota, may account for these N2 balance differences. To investigate this, we correlated gut microbial profiles with the growth of four murine strains (C57Bl6/J, CD-1, FVB, and NIH-Swiss) on protein deficient (PD) diet. Results show that a PD diet exerts a strain-dependent impact on growth and N2 balance as determined through analysis of urinary urea, ammonia and creatinine excretion. Bacterial alpha diversity was significantly (p < 0.05, FDR) lower across all strains on a PD diet compared to normal chow (NC). Multi-group analyses of the composition of microbiomes (ANCOM) revealed significantly differential microbial signatures between the four strains independent of diet. However, mice on a PD diet demonstrated differential enrichment of bacterial genera including, Allobaculum (C57Bl6/J), Parabacteroides (CD-1), Turicibacter (FVB), and Mucispirillum (NIH-Swiss) relative to NC. Additionally, statistical model fitting revealed that the relative abundance of genera such as Bifidobacterium, Ruminococcus, and Lactobacillus were significantly positively correlated with body weight, while Anaerofustis, Roseburia, and Bilophila were significantly positively correlated with ammonia excretion. Taken together, these results suggest a potential relationship between the specific gut microbiota, N2 balance and animal response to malnutrition.

A Fully Automated, Faster Noise Reduction Approach to Increasing the Analytical Capability of Chemical Imaging for Digital Histopathology

Gupta, S. — 2018-09-24

High dimensional data, for example from infrared spectral imaging, involves an inherent trade-off in the acquisition time and quality of spatial-spectral data. Minimum Noise Fraction (MNF) developed by Green et al. [1] has been extensively studied as an algorithm for noise removal in HSI (Hyper-Spectral Imaging) data. However, there is a speed-accuracy trade-off in the process of manually deciding the relevant bands in the MNF space, which by current methods could become a person month time for analyzing an entire TMA (Tissue Micro Array). We propose three approaches termed Fast MNF, Approx MNF and Rand MNF where the computational time of the algorithm is reduced, as well as the entire process of band selection is fully automated. This automated approach is shown to perform at the same level of reconstruction accuracy as MNF with large speedup factors, resulting in the same task to be accomplished in hours. The different approximations of the algorithm, show the reconstruction accuracy vs storage (50x) and runtime speed (60x) trade-off. We apply the approach for automating the denoising of different tissue histology samples, in which the accuracy of classification (differentiating between the different histologic and pathologic classes) strongly depends on the SNR (signal to noise ratio) of recovered data. Therefore, we also compare the effect of the proposed denoising algorithms on classification accuracy. Since denoising HSI data is done without any ground truth, we also use a metric that assesses the quality of denoising in the image domain between the noisy and denoised image in absence of ground truth.

Commensal gut bacteria convert the immunosuppressant tacrolimus to less potent metabolites

Guo, Y. — 2018-09-26

Tacrolimus exhibits low and variable drug exposure after oral dosing, but the contributing factors remain unclear. Based on our recent report showing a positive correlation between fecal abundance of Faecalibacterium prausnitzii and oral tacrolimus dose in kidney transplant patients, we tested whether F. prausnitzii and other gut abundant bacteria are capable of metabolizing tacrolimus. Incubation of F. prausnitzii with tacrolimus led to production of two compounds (the major one named M1), which was not observed upon tacrolimus incubation with hepatic microsomes. Isolation, purification, and structure elucidation using mass spectrometry and nuclear magnetic resonance spectroscopy indicated that M1 is a C-9 keto-reduction product of tacrolimus. Pharmacological activity testing using human peripheral blood mononuclear cells demonstrated that M1 is 15-fold less potent than tacrolimus as an immunosuppressant. Screening of 22 gut bacteria species revealed that most Clostridiales bacteria are extensive tacrolimus metabolizers. Tacrolimus conversion to M1 was verified in fresh stool samples from two healthy adults. M1 was also detected in the stool samples from kidney transplant recipients who had been taking tacrolimus orally. Together, this study presents gut bacteria metabolism as a previously unrecognized elimination route of tacrolimus, potentially contributing to the low and variable tacrolimus exposure after oral dosing.

Human APOBEC3G prevents emergence of infectious endogenous retrovirus in mice

Treger, R. — 2018-10-31

Endogenous retroviruses (ERV) are found throughout vertebrate genomes and failure to silence their activation can have deleterious consequences on the host. Introduction of mutations that subsequently prevent transcription of ERV loci is therefore an indispensable cell-intrinsic defense mechanism that maintains the integrity of the host genome. Abundant in vitro and in silico evidence have revealed that APOBEC3 cytidine-deaminases, including human APOBEC3G (hA3G) can potently restrict retrotransposition; yet in vivo data demonstrating such activity is lacking, particularly since no replication competent human ERV has been identified. In mice deficient for Toll-like receptor 7 (TLR7), transcribed ERV loci can recombine and generate infectious ERV. In this study, we show that mice deficient in the only copy of Apobec3 in the genome did not have spontaneous reactivation of ERVs, nor elevated ERV reactivation when crossed to Tlr7-/- mice. In contrast, expression of a human APOBEC3G transgene abrogated emergence of infectious ERV in the Tlr7-/- background. No ERV RNA was detected in the plasma of hA3G+Apobec3-/-Tlr7-/- mice, and infectious ERV virions could not be amplified through co-culture with permissive cells. These data reveal that hA3G can potently restrict active ERV in vivo, and suggest that the expansion of the APOBEC3 locus in primates has helped restrict ERV reactivation in the human genome.nnImportanceAlthough APOBEC3 proteins are known to be important antiviral restriction factors in both mice and humans, their roles in the restriction of endogenous retroviruses (ERV) have been limited to in vitro studies. Here, we report that human APOBEC3G expressed as a transgene in mice prevents the emergence of infectious ERV from endogenous loci. This study reveals that APOBEC3G can powerfully restrict active retrotransposons in vivo and demonstrates how ectopic expression of human factors in transgenic mouse models can be used to investigate host mechanisms that inhibit retrotransposons and reinforce genomic integrity.

Organ transformation by environmental disruption of epigenetic memory

Snir, O. — 2018-11-29

Despite major progress in mechanistic understanding of epigenetic reprogramming of cells, the basis of organ reprograming by (epi-)gene-environment interactions remained largely obscured. Here we use the ether-induced haltere-to-wing transformations as a model for epigenetic "reprogramming" at the whole organism level. Our findings support a mechanistic chain of events explaining why and how brief embryonic exposure to ether leads to organ transformation manifested at the larval stage and on. We show that ether interferes with protein integrity in the egg leading to altered deployment of Hsp90 and repression of Trithorax-mediated establishment of H3K4 tri-methylations. This repression pre-disposes early methylated Ubx targets and wing genes for later up-regulation in the larval haltere disc, hence the wing-like outcome. Consistent with compromised protein integrity during the exposure, the severity of bithorax transformation is increased by genetic or chemical reduction of Hsp90 function. Moreover, a joint reduction in Hsp90 and trx gene dosage can cause bithorax transformations without exposure to ether. These findings implicate environmental disruption of protein integrity at the onset of histone methylations with a modification of epigenetic memory, which in turn, supports a morphogenetic shift towards an ancestral-like body plan. The morphogenetic impact of chaperone response during a major setup of epigenetic patterns may be a general scheme for organ reprogramming by environmental cues.

Novel genes for autism implicate both excitatory and inhibitory cell lineages in risk

Satterstrom, F. K. — 2018-11-30

We present the largest exome sequencing study to date focused on rare variation in autism spectrum disorder (ASD) (n=35,584). Integrating de novo and case-control variation with an enhanced Bayesian framework incorporating evolutionary constraint against mutation, we implicate 99 genes in ASD risk at a false discovery rate (FDR) [≤] 0.1. Of these 99 risk genes, 46 show higher frequencies of disruptive de novo variants in individuals ascertained for severe neurodevelopmental delay, while 50 show higher frequencies in individuals ascertained for ASD, and comparing ASD cases with disruptive mutations in the two groups shows differences in phenotypic presentation. Expressed early in brain development, most of the risk genes have roles in neuronal communication or regulation of gene expression, and 12 fall within recurrent copy number variant loci. In human cortex single-cell gene expression data, expression of the 99 risk genes is also enriched in both excitatory and inhibitory neuronal lineages, implying that disruption of these genes alters the development of both neuron types. Together, these insights broaden our understanding of the neurobiology of ASD.

UNC-16/JIP3 inhibits the function of the regeneration promoting isoform of DLK-1

Kulkarni, S. S. — 2018-12-02

Neuronal regeneration after injury depends on the intrinsic growth potential of neurons. UNC-16, a C. elegans JIP3 homologue, inhibits axonal regeneration by regulating regrowth initiation and rate of regrowth. UNC-16/JIP3 inhibits the regeneration promoting activity of DLK-1 long but acts additively to and independently of inhibitory DLK-1 short isoform. UNC-16/JIP3 promotes DLK-1 punctate localization in a concentration dependent manner limiting DLK-1 long availability at the cut site minutes after injury. UNC-16 negatively regulates actin dynamics dependent on DLK-1 and microtubule dynamics independent of DLK-1. The faster regeneration seen in unc-16 does not lead to functional recovery. We propose a model where UNC-16/JIP3 plays its inhibitory role through tight temporal and spatial control of DLK-1 function. The dual inhibitory control by both UNC-16 and DLK-1 short calibrate the intrinsic growth promoting function of DLK-1 long in vivo.

Identifying small molecule probes of ENTPD5 through high throughput screening

Durst, M. A. — 2018-12-21

Ectonucleoside Triphosphate Diphosphohydrolase 5 (ENTPD5) has been shown to be important in maintaining cellular function in cancer, and its expression is upregulated through multiple, unique pathways in certain cancers, including laryngeal, glioblastoma multiforme, breast, testicular, and prostate. ENTPD5 supports cancer growth by promoting the import of UDP-glucose, a metabolite used for protein glycosylation and hence proper glycoprotein folding, into the ER by providing the counter molecule, UMP, to the ER antiporter. Despite its cancer-supporting function, no small molecule inhibitors of ENTPD5 are commercially available, and few studies have been performed in tissue culture to understand the effects of chemical inhibition of ENTPD5. We performed a high-throughput screen (HTS) of 21,120 compounds to identify small molecule inhibitors of ENPTD5 activity. Two hits were identified, and we performed a structure activity relationship (SAR) screen around these hits. Further validation of these probes were done in an orthogonal assay and then assayed in cell culture to assess their effect on prostate cancer cell lines. Notably, treatment with the novel ENTPD5 inhibitor reduced the amount of glycoprotein produced in treated cells, consistent with the hypothesis that ENTPD5 is important for glycoprotein folding. This work serves as an important step in designing new molecular probes for ENTPD5 as well as further probing the utility of targeting ENTPD5 to combat cancer cell proliferation.

Perineuronal Nets in the Insula Regulate Aversion-Resistant Alcohol Drinking

Chen, H. — 2018-12-21

One of the most pernicious characteristics of alcohol use disorder is the compulsion to drink despite negative consequences. The insular cortex (insula) controls decision-making under conditions of risk or conflict and regulates maladaptive behaviors in the context of addiction. Cortical activity is tightly controlled by fast-spiking inhibitory interneurons that are often enclosed by specialized extracellular matrix structures known as perineuronal nets, which regulate neuronal excitability and plasticity. Using a mouse model of compulsive drinking in which alcohol was adulterated with the bitter tastant quinine, we demonstrate that disrupting perineuronal nets in the insula rendered mice more sensitive to quinine-adulterated alcohol. Activation of the insula, as measured by c-fos expression, occurred during aversion-resistant drinking and was further enhanced by elimination of perineuronal nets. These results provide fundamental insight into neuroanatomical and cellular mechanisms that control compulsive drinking.

Quantitative Analysis of Global Protein Stability Rates in Tissues

McClatchy, D. B. — 2019-01-15

Protein degradation is an essential mechanism for maintaining homeostasis in response to internal and external perturbations. Disruption of this process is implicated in many human diseases, but quantitation of global stability rates has not yet been achieved in tissues. We have developed QUAD (Quantification of Azidohomoalanine Degradation), a technique to quantitate global protein degradation using mass spectrometry. Azidohomoalanine (AHA) is pulsed into mouse tissues through their diet. The mice are then returned to a normal diet and the decrease of AHA abundance can be quantitated in the proteome. QUAD analysis reveals that protein stability varied within tissues, but discernible trends in the data suggest that cellular environment is a major factor dictating stability. Within a tissue, different organelles, post-translation modifications, and protein functions were enriched with different stability patterns. Surprisingly, subunits of the TRIC molecular chaperonin possessed markedly distinct stability trajectories in the brain. Further investigation revealed that these subunits also possessed different subcellular localization and expression patterns that were uniquely altered with age and in Alzheimers disease transgenic mice, indicating a potential non-canonical chaperonin. Finally, QUAD analysis demonstrated that protein stability is enhanced with age in the brain but not in the liver. Overall, QUAD allows the first global quantitation of protein stability rates in tissues, which may lead to new insights and hypotheses in basic and translational research.nnSummaryProtein degradation is an important component of the proteostasis network, but no techniques are available to globally quantitate degradation rates in tissues. In this study, we demonstrate a new method QUAD (Quantification of Azidohomoalanine Degradation) that can accurately quantitate degradation rates in tissues. QUAD analysis of mouse tissues reveal that unique degradation trends can define different tissue proteomes. Within a tissue, specific protein characteristics are correlated with different levels of protein stability. Further investigation of the TRIC chaperonin with strikingly different subunit stabilities suggests a non-canonical chaperonin in brain tissue. Consistent with the theory that the proteostasis network is compromised with age, we discovered that protein stability is globally enhanced in brains of old mice compared to young mice.

Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

Meydan, S. — 2019-01-16

The use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes but, strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the alternative bacterial proteome. The internal start sites my also play regulatory roles in gene expression.

METABOLIC DIVERSITY IN HUMAN NON-SMALL CELL LUNG CANCER CELLS

CHEN, P.-H. — 2019-03-11

Intermediary metabolism in cancer cells is regulated by diverse cell-autonomous processes including signal transduction and gene expression patterns arising from specific oncogenotypes and cell lineages. Although it is well established that metabolic reprogramming is a hallmark of cancer, we lack a full view of the diversity of metabolic programs in cancer cells and an unbiased assessment of the associations between metabolic pathway preferences and other cell-autonomous processes. Here we quantified over 100 metabolic features, mostly from 13C enrichment of molecules from central carbon metabolism, in over 80 non-small cell lung cancer (NSCLC) cell lines cultured under identical conditions. Because these cell lines were extensively annotated for oncogenotype, gene expression, protein expression and therapeutic sensitivity, the resulting database enables the user to uncover new relationships between metabolism and these orthogonal processes.

Combinatorial morphogenetic and mechanical cues to mimic bone development for defect repair

Herberg, S. — 2019-02-26

Endochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation and is directed by local morphogen signals and mechanical cues. Here, we aimed to mimic these developmental conditions for regeneration of large bone defects. We hypothesized that engineered human mesenchymal stem cell (hMSC) condensations with in situ presentation of transforming growth factor-{beta}1 (TGF-{beta}1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles would promote endochondral regeneration of critical-sized rat femoral bone defects in a manner dependent on the in vivo mechanical environment. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with dual BMP-2 + TGF-{beta}1 fully restoring mechanical bone function by week 12. In vivo ambulatory mechanical loading, initiated at week 4 by delayed unlocking of compliant fixation plates, significantly enhanced the bone formation rate in the four weeks after load initiation in the dual morphogen group. In vitro, local presentation of either BMP-2 alone or BMP-2 + TGF-{beta}1 initiated endochondral lineage commitment of mesenchymal condensations, inducing both chondrogenic and osteogenic gene expression through SMAD3 and SMAD5 signaling. In vivo, however, endochondral cartilage formation was evident only in the BMP-2 + TGF-{beta}1 group and was enhanced by mechanical loading. The degree of bone formation was comparable to BMP-2 soaked on collagen but without the ectopic bone formation that limits the clinical efficacy of BMP-2/collagen. In contrast, mechanical loading had no effect on autograft-mediated repair. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering. One Sentence SummaryMimicking aspects of the cellular, biochemical, and mechanical environment during early limb development, chondrogenically-primed human mesenchymal stem cell condensations promoted functional healing of critical-sized femoral defects via endochondral ossification, and healing rate and extent was a function of the in vivo mechanical environment.

Crosstalk with the GAR-3 receptor contributes to feeding defects in Caenorhabditis elegans eat-2 mutants

Kozlova, A. — 2019-02-26

Precise signaling at the neuromuscular junction (NMJ) is essential for proper muscle contraction. In the C. elegans pharynx, acetylcholine (ACh) released from the MC and M4 motor neurons stimulates two different types of contractions in adjacent muscle cells, termed pumping and isthmus peristalsis. MC stimulates rapid pumping through the nicotinic ACh receptor EAT-2, which is tightly localized at the MC NMJ, and eat-2 mutants exhibit a slow pump rate. Surprisingly, we found that eat-2 mutants also hyperstimulated peristaltic contractions, and these are characterized by increased and prolonged Ca2+ transients in the isthmus muscles. This hyperstimulation depends on crosstalk with the GAR-3 muscarinic acetylcholine receptor as gar-3 mutation specifically suppressed the prolonged contraction and increased Ca2+ observed in eat-2 mutant peristalses. Similar GAR-3 dependent hyperstimulation was also observed in mutants lacking the ace-3 acetylcholinesterase, and we suggest that NMJ defects in eat-2 and ace-3 mutants result in ACh stimulation of extrasynaptic GAR-3 receptors in isthmus muscles. gar-3 mutation also suppressed slow larval growth and prolonged lifespan phenotypes that result from dietary restriction in eat-2 mutants, indicating that crosstalk with the GAR-3 receptor has a long-term impact on feeding behavior and eat-2 mutant phenotypes. Article SummaryAcetylcholine stimulates different contractions in adjacent muscle cells in the C. elegans pharynx called pumping and peristalsis. The signaling mechanisms stimulating pumping have been characterized, but how these mechanisms affect peristalsis is unknown. Here we examined muscle contractions and Ca2+ transients during peristalsis in wild-type animals and acetylcholine signaling mutants. Surprisingly we found that while mutants affecting the eat-2 nicotinic acetylcholine receptor exhibited reduced pumping, they also hyperstimulated peristalses. This hyperstimulation depends on crosstalk with the GAR-3 muscarinic acetylcholine receptor in adjacent cells, and it contributes to the well-characterized dietary restriction and extended adult lifespan observed in eat-2 mutants.

Syndecan-1 facilitates breast cancer metastasis to the brain

Sayyad, M. R. — 2019-03-02

PurposeAlthough survival rates for patients with localized breast cancer have increased, patients with metastatic breast cancer still have poor prognosis. Understanding key factors involved in promoting breast cancer metastasis is imperative for better treatments. In this study, we investigated the role of syndecan-1 (Sdc1) in breast cancer metastasis. MethodsTo assess the role of Sdc1 in breast cancer metastasis, we silenced Sdc1 expression in the triple-negative breast cancer human MDA-MB-231 cell line and overexpressed it in the mouse mammary carcinoma 4T1 cell line. Intracardiac injections were performed in an experimental mouse metastasis model using both cell lines. In vitro transwell blood-brain barrier (BBB) and brain section adhesion assays were utilized to specifically investigate how Sdc1 promotes brain metastasis. A cytokine array was performed to evaluate differences in the breast cancer cell secretome when Sdc1 was silenced. ResultsSilencing expression of Sdc1 in breast cancer cells significantly reduced metastasis to the brain. Conversely, overexpression of Sdc1 increased metastasis to the brain. We found that the reduction in brain metastases with Sdc1 knockdown was likely due to reduced breast cancer cell migration across the BBB and adhesion to the perivascular regions of the brain. However, there was no change in attachment to brain endothelial cells or astrocytes. Loss of Sdc1 also led to changes in breast cancer cell-secreted cytokines, which may influence the BBB. ConclusionsTaken together, our study demonstrates a role for Sdc1 in promoting breast cancer metastasis to the brain. These findings suggest that Sdc1 supports breast cancer cell migration across the BBB through regulation of cytokines, which may modulate the BBB. Further elucidating this mechanism will allow for the development of therapeutic strategies to combat brain metastasis.

Prevalence of parasitic infections among recent immigrants to Chicago

Herrick, J. A. — 2019-03-13

BackgroundParasitic infections are likely under-recognized among immigrant populations in the United States (US). We conducted a cross-sectional study to evaluate the frequency of such infections among recent immigrants in Chicago and to identify predictive factors for parasitic infections.nnMethodology and principal findings133 recent immigrants were enrolled, filling out a standardized questionnaire regarding medical history and exposures and providing blood and stool samples for evaluation. Fifteen of 125 subjects (12%) who provided a blood or stool sample for testing were found to have evidence of current or prior infection with a pathogenic parasite, of which Toxocara spp. (8 subjects, 6.4%) and Strongyloides stercoralis (5 subjects, 4%) were most commonly identified. Parasitic infection was more likely among subjects who had immigrated within the previous 2 years and those with a self-reported history of ever having seen worms in the stool. Infected individuals were likely to have multiple nonspecific physical complaints; however, classic symptoms of parasitic infections (skin rashes, diarrhea, etc.) were not increased among infected individuals. The most useful surrogate markers identified for parasitic infections were an elevated Immunoglobulin E level (seen in 7/15 subjects with parasitic infections, 46.7% and 22/110 uninfected individuals, 20%, p=0.04) and the presence of Blastocystis hominis cysts on Ova & Parasite exam (detected in 5/13 subjects with parasitic infections who provided a stool sample, 38.5% and 5/98 uninfected subjects, 5.1%, p=0.002). In contrast, the Absolute Eosinophil Count (typically thought of as an indicator of parasites) was not found to be a good screening test for parasitic infections in this study.nnConclusionsOur study found that parasitic infections are common in recent US immigrants, which highlights an important health disparity among a vulnerable population. Further, we found that classically used symptoms and laboratory tests had a low predictive value for parasitic infections in this population.nnAUTHOR SUMMARYParasitic infections, though rare in the United States (US), are common in many areas of the world including the regions of origin of many US immigrants. However, the prevalence rates and health impacts of these infections in immigrant populations are undefined. We conducted a study to identify the frequency of parasitic infections among healthy immigrants in one community, recruiting 133 immigrants from 28 countries. Subjects completed a standardized questionnaire regarding symptoms and infection risk-factors and provided blood and stool samples for testing. Twelve percent of subjects in our study had evidence of current or previous pathogenic parasitic infections. Symptoms and risk factors classically thought to be associated with parasitic infection (allergic symptoms, elevated blood eosinophil counts, etc.) were common among enrolled subjects, but did not differ significantly between those with and without evidence for infection. Overall, our results suggest that many immigrants, even those who are asymptomatic, may have undiagnosed parasitic infections. These results highlight an important health disparity among a vulnerable underserved population in the US. As most of these infections are easily treatable, more research should be done to further characterize the optimal testing strategies for recent immigrants.

Higher order analysis of gene correlations by tensor decomposition

Yahyanejad, F. — 2019-03-16

This study advances our understanding of inter- and intra-pathways higher order signaling in the cellular system and it leads to new discovery of multiple intracellular structures in signal transduction pathways in yeast Saccharomyces. We present a new tensor decomposition algorithm in reconstructing the pathways based on higher correlations among genes that compose a cellular system. The higher order gene correlation (HOGC) analysis has the power to elucidate genes higher interaction dependencies which has been barely understood. Recent studies i.e. [24] have experimentally revealed that multiple signaling proteins, yet sometimes infinite, may assemble to meaningful structure to transmit a receptor activation information. In this paper we reveal 3-order genomic correlations among significant component of the cellular system. This is the first time such a systematic and computational model provided for analysis of higher order correlations among genes. We use new fast algorithm to formulate a genes x genes x genes x decorrelated rank-1 sub-tensors (complexes) which can be associated with functionally independent pathways. Then we model higher order tensor decomposition [Formula] which is constructed by K tensors of genes x genes x genes. Each new tensor is constructed by an orthogonal projection of data signal onto a designated basis signal to keep common sub-tensors in both signals. Our model for decomposing tensor order-4 approximates series of tensors as linear components of deccorelated rank-1 sub-tensors over tensor of order-3 and rank-3 triplings among sub-tensors. The linear components represent intra-pathway in cell signaling and triplings implicate inter-pathways higher order signaling. Through structural studies of inter- and intra-higher order signaling pathways, we uncover different scenario that involves triple formation of signaling proteins into higher order signaling machines for transmission of receptor activation information to cellular responses.

HAVE IT, KNOW IT, BUT DO NOT SHOW IT: EXAMINING PHYSIOLOGICAL AROUSAL, ANXIETY, AND FACIAL EXPRESSIONS OVER THE COURSE OF A SOCIAL SKILLS INTERVENTION FOR AUTISTIC ADOLESCENTS

Jain, N. — 2019-03-25

Facial expressions provide a nonverbal mechanism for social communication, a core challenge for autistic people. Little is known regarding the association between arousal, self-report of anxiety, and facial expressions among autistic adolescents. Therefore, this study investigated session-by-session facial expressions, self-report of anxiety, and physiological arousal via Electrodermal Activity (EDA), of 12 autistic male adolescents in a didactic social skills intervention setting. The goals of this study were threefold: 1) identify physiological arousal levels ("have-it"), 2) examine if autistic adolescents facial expressions indicated arousal ("show-it"), and 3) determine whether autistic adolescents were self-aware of their anxiety ("know-it"). Our results showed that autistic adolescents self-rated anxiety was significantly associated with peaks in EDA. Both machine learning algorithms and human participant-based methods, however, had low accuracy in predicting autistic adolescents arousal state from facial expressions, suggesting that autistic adolescents facial expressions did not coincide with their arousal. Implications for understanding social communication difficulties among autistic adolescents, as well as future targets for intervention, are discussed. This project is registered with ClinicalTrials.gov, Identifier: NCT02680015.

Rule-governed Dynamic Stochastic Equilibration of Multicellular Motion In Vivo During Olfactory Neurogenesis

Warrier, V. — 2019-03-28

The complexity of patterning during organ-wide stem cell migration and differentiation can be challenging to interpret quantitatively. Here, we track neural crest (NC) and ectodermal placode-derived progenitor movements in vivo, for hundreds of cells, implement unbiased algorithmic approaches to extract biologically meaningful information, and discover cell-cell and lineage-lineage coordination between progenitors that form olfactory sensory neurons (OSNs) during zebrafish embryogenesis. Our approach discriminates between NC- and placode-derived contributions and segregates ingressing NC cells into two previously unidentified subtypes termed trend and dispersed lineages. Our analyses indicate that NC and placodal progenitor migration and intercalation are coordinated by at least two types of collective behavior: spatiotemporal exclusion and elastic tethering, akin to a push-pull mechanism. A stochastic equilibrium model accurately represents the interactions of NC and placode-derived lineages. Our approach provides insights into the coordination of dual-origin lineages during vertebrate olfactory neurogenesis and offers an algorithmic toolkit for probing multicellular coordination in vivo.nnO_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Graphical Abstract C_FIG_DISPLAY

The human microbiota is associated with cardiometabolic risk across the epidemiologic transition

Fei, N. — 2019-04-01

Oral and fecal microbial biomarkers have previously been associated with cardiometabolic (CM) risk, however, no comprehensive attempt has been made to explore this association in minority populations or across different geographic regions. We characterized gut- and oral-associated microbiota and CM risk in 655 participants of African-origin, aged 25-45, from Ghana, South Africa, Jamaica, and the United States (US). CM risk was classified using the CM risk cut-points for elevated waist circumference, elevated blood pressure and elevated fasted blood glucose, low high-density lipoprotein (HDL), and elevated triglycerides. Gut-associated bacterial alpha diversity negatively correlated with elevated blood pressure and elevated fasted blood glucose. Similarly, gut bacterial beta diversity was also significantly differentiated by waist circumference, blood pressure, triglyceridemia and HDL-cholesterolemia. Notably, differences in inter- and intra-personal gut microbial diversity were geographic-region specific. Participants meeting the cut-points for 3 out of the 5 CM risk factors were significantly more enriched with Lachnospiraceae, and were significantly depleted of Clostridiaceae, Peptostreptococcaceae, and Prevotella. The predicted relative proportions of the genes involved in the pathways for lipopolysaccharides (LPS) and butyrate synthesis were also significantly differentiated by the CM risk phenotype, whereby genes involved in the butyrate synthesis via lysine, glutarate and 4-aminobutyrate/succinate pathways and LPS synthesis pathway were enriched in participants with greater CM risk. Furthermore, inter-individual oral microbiota diversity was also significantly associated with the CM risk factors, and oral-associated Streptococcus, Prevotella, and Veillonella were enriched in participants with 3 out of the 5 CM risk factors. We demonstrate that in a diverse cohort of African-origin adults, CM risk is significantly associated with reduced microbial diversity, and the enrichment of specific bacterial taxa and predicted functional traits in both gut and oral environments. As well as providing new insights into the associations between the gut and oral microbiota and CM risk, this study also highlights the potential for novel therapeutic discoveries which target the oral and gut microbiota in CM risk.

Anti-HIV-1 Activity of Crocodylus mindorensis (Philippine crocodile) serum in cell-free and cell-associated virus interactions to human peripheral blood mononuclear cells

Hinay, A. A. — 2019-04-04

Highly-Active Antiretroviral Therapy (HAART) is the recommended treatment and management strategy for HIV infection. Although the existing antiretroviral drugs are indispensably significant in improving the quality of lives of HIV/AIDS individuals, the drugs still have many limitations including resistance, production of toxicity, and their limited availability. These limitations continue to open new opportunities in the use of ethnomedicine for the management of HIV/AIDS. With this, few researchers have made an effort to test the inhibitory activity of crocodile serum as it has a unique and diverse molecular activity in preventing HIV-1 replication. In this study, a cell culture-based assay was utilized coupled with colorimetric enzyme immunoassay to determine the HIV-1 reverse transcriptase activity. One HIV-1 seropositive serum was processed for Peripheral Blood Mononuclear Cells (PBMC) co-culture from which HIV-1 isolates were obtained. The HIV-1 reverse transcriptase activity after 21 days was 0.5928 pg/well. Moreover, a baseline Philippine crocodile serum concentration of 0.5% vol/vol was used based on the previous study conducted by Hinay and Sarol (2018) and the cell viability results showed no cell reduction of mononuclear cells after 72 hours incubation. The inhibitory activity of the Philippine crocodile serum at 0.5% and 0.25% vol/vol concentrations inhibited 65.68{+/-}2.93% and 69.92{+/-}0.45% respectively in post-infection interactions. In addition, the Philippine crocodile serum in pre-infection interaction at 0.5% and 0.25% vol/vol concentrations inhibited 68.61{+/-}1.67% and 69.95{+/-}2.24% respectively. As has been noted, the inhibitory actions of the Philippine crocodile serum effectively regulate the HIV-1 replication in both pre- and post-infection interactions.

Early Detection of Ovarian Cancer Using Cells Sourced from a Local Microenvironment

Galey, M. M. — 2019-04-04

Mass spectrometry (MS) offers high levels of specificity and sensitivity in clinical applications, and we have previously been able to demonstrate that matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS is capable of distinguishing two-component biological mixtures at low limits of detection. Ovarian cancer is notoriously difficult to detect due to the lack of any screening methods for early detection. By sampling a local microenvironment, such as the vaginal fluids, a MS based method is presented that was capable of monitoring disease progression from vaginally collected, local samples from tumor bearing mice. A murine xenograft model of high grade serous ovarian carcinoma (HGSOC) was used for this study and vaginal lavages were obtained from mice on a weekly basis throughout disease progression and subjected to our MALDI-TOF MS workflow followed by statistical analyses. Proteins in the 4-20 kDa region of the mass spectrum could consistently be measured to yield a fingerprint that correlated with disease progression over time. These fingerprints were found to be statistically stable across all mice with the protein fingerprint converging towards the end point of the study. MALDI-TOF MS serves as a unique analytical technique for measuring a sampled vaginal microenvironment in a specific and sensitive manner for the detection of HGSOC in a murine model.

Living cell-only bioink and photocurable supporting medium for printing and generation of engineered tissues with complex geometries

Jeon, O. — 2019-04-17

Scaffold-free engineering of three-dimensional (3D) tissue has focused on building sophisticated structures to achieve functional constructs. Although the development of advanced manufacturing techniques such as 3D printing has brought remarkable capabilities to the field of tissue engineering, technology to create and culture individual cell only-based high-resolution tissues, without an intervening biomaterial scaffold to maintain construct shape and architecture, has been unachievable to date. In this report, we introduce a cell printing platform which addresses the aforementioned challenge and permits 3D printing and long-term culture of a living cell-only bioink lacking a biomaterial carrier for functional tissue formation. A biodegradable and photocrosslinkable microgel supporting bath serves initially as a fluid, allowing free movement of the printing nozzle for high-resolution cell extrusion, while also presenting solid-like properties to sustain the structure of the printed constructs. The printed human stem cells, which are the only component of the bioink, couple together via transmembrane adhesion proteins and differentiate down tissue-specific lineages while being cultured in a further photocrosslinked supporting bath to form bone and cartilage tissue with precisely controlled structure. Collectively, this system, which is applicable to general 3D printing strategies, is paradigm shifting for printing of scaffold-free individual cells, cellular condensations and organoids, and may have far reaching impact in the fields of regenerative medicine, drug screening, and developmental biology.

EphB1 in Endothelial Cells Regulates Caveolae Formation and Endocytosis

Tiruppathi, C. — 2019-04-26

Caveolae, the cave-like structures abundant in endothelial cells (ECs), are important in regulating key functions such as caveolae-mediated endocytosis and generation of nitric oxide. Here we show that deletion of the receptor tyrosine kinase EphB1 (EphB1-/-) in mice markedly reduced the caveolae number in ECs of heart and lung vessels and prevented caveolae-mediated endocytosis. EphB1 expressed in adult ECs was shown to bind the caveolin-1 (Cav-1) scaffold domain (CSD) via the CSD binding motif (CSDBM) on EphB1. We demonstrated that activation of EphB1 by the native ligand Ephrin B1 uncoupled EphB1 from Cav-1, and licensed Src-dependent Y-14 Cav-1 phosphorylation. Deletion of CSDBM on EphB1 prevented EphB1/Cav-1 interaction and the activation of Src and Src mediated Y-14 Cav-1 phosphorylation. These studies identify the central role of endothelium expressed EphB1 in regulating caveolae biogenesis and caveolae-mediated endocytosis.

Corticothalamic gating of population auditory thalamocortical transmission in mouse

Ibrahim, B. A. — 2019-05-03

Since the discovery of the receptive field, scientists have tracked receptive field structure to gain insights about mechanisms of sensory processing. At the level of the thalamus and cortex, this linear filter approach has been challenged by findings that populations of cortical neurons respond in a stereotyped fashion to sensory stimuli. Here, we elucidate a possible mechanism by which gating of cortical representations occurs. All-or-none population responses (here called "ON" and "OFF" responses) were observed in vivo and in vitro in the mouse auditory cortex at near-threshold acoustic or electrical stimulation. ON-responses were associated with previously-described UP states in the auditory cortex. OFF-responses in the cortex were only eliminated by blocking GABAergic inhibition in the thalamus. Opto- and chemogenetic silencing of NTSR-positive corticothalamic layer 6 (CTL6) neurons as well as the pharmacological blocking of the thalamic reticular nucleus (TRN) retrieved the missing cortical responses, suggesting that the corticothalamic feedback inhibition via TRN controls the gating of thalamocortical activity. Moreover, the oscillation of the pre-stimulus activity of corticothalamic cells predicted the cortical ON vs. OFF responses, suggesting that underlying cortical oscillation controls thalamocortical gating. These data suggest that the thalamus may recruit cortical ensembles rather than linearly encoding ascending stimuli and that corticothalamic projections play a key role in selecting cortical ensembles for activation.

Ultrafast Structural Changes Decomposed from Serial Crystallographic Data

Ren, Z. — 2019-05-10

Direct visualization of electronic and molecular events during chemical and biochemical reactions will offer unprecedented mechanistic insights. Ultrashort pulses produced by X-ray free electron lasers (XFELs) offer an unprecedented opportunity for direct observations of transient events as short-lived as tens of femtoseconds. This paper presents an in-depth analysis of the serial crystallographic datasets collected by Barends & Schlichting et al. (Science 350, 445, 2015) that probe the ligand photodissociation in carbonmonoxy myoglobin (MbCO), a long-serving hallmark for observing ultrafast dynamics in a biological system. This analysis reveals electron density changes that are caused directly by the formation of high-spin 3d atomic orbitals of the heme iron upon the CO departure and the dynamic behaviors of these newly formed orbitals in a time series within the first few picoseconds. The heme iron is found vibrating at a high frequency and developing a positional modulation that causes the iron to pop out of and recoil back into the heme plane in succession. These findings here provide long-awaited visual validations for previous works using ultrafast spectroscopy and molecular dynamics simulations. This analysis also extracts electron density variations largely in the solvent during the first period of a low frequency oscillation previously detected by coherence spectroscopy. This work demonstrates the power and importance of the analytical methods in detecting and isolating transient, often very weak signals of electronic changes arising from chemical reactions in proteins.nnSummaryDirect imaging of ultrafast and subtle structural events during a biochemical reaction, such as a single electronic transition from one atomic or molecular orbital to another, is highly desirable but has been beyond the reach of protein crystallography. It entails the capability of observing changes in electronic distributions at both an ultrafast time scale and an ultrahigh spatial resolution (Itatani et al., Nature 432, 867, 2004). The recent developments in femtosecond serial crystallography at X-ray free electron lasers (XFELs) have brought the achievable temporal resolution within a striking distance. This paper presents the electron density map decomposed from the XFEL data that shows the remanence of several 3d atomic orbitals of the heme iron at an available spatial resolution although the map component is not an accurate image of the atomic orbitals. A key strategy that has enabled the findings here is a numerical deconvolution to resolve concurrent variations in a series of time-resolved electron density maps so that the electron densities influenced by an electron transfer event can be isolated as a partial change from the overwhelming presence of the bulk electrons that are not directly involved in bonding. Even at the limited spatial resolution, the subtle changes in electron distribution due to a spin crossover can be decoupled from far greater changes due to atomic displacements. Direct observations of electronic orbitals could offer unprecedented mechanistic insights into a myriad of chemical and biochemical reactions such as electron transfer in redox reactions, and formation, rupture, and isomerization of chemical bonds.nnLigand photodissociation in carbonmonoxy myoglobin (MbCO) has been a benchmark for studying ultrafast protein dynamics in a biological system. A number of studies in time-resolved crystallography have progressively improved the time resolution (from [S]rajer et al., Science 274, 1726, 1996 to Barends et al., Science 350, 445, 2015). This paper presents an in-depth analysis of the serial crystallographic datasets of MbCO that Barends & Schlichting et al. (2015) contributed to the Protein Data Bank. First, a component of electron density distributions clearly shows the characteristic shape of the high-spin 3d orbitals reappeared at the heme iron upon the photodissociation of the CO ligand despite the limited accuracy of the orbital image due to the available spatial resolution. Second, the dynamic behaviors of these newly regained 3d orbitals within picoseconds after the photolysis provide long-awaited structural validation for previous spectroscopic observations and computational simulations. Specifically, the newly formed densities are oscillating with the heme iron at a high frequency of a thousand wavenumbers and developing a positional modulation during the first few picoseconds (Champion, Science 310, 980, 2005). The iron pops out of the heme plane at a few picoseconds and recoils back and pops out again afterwards. The dominant oscillation at a low frequency of several tens wavenumbers previously detected by coherence spectroscopy can be clearly resolved from the time series of electron density maps. The associated changes in electron density during the first cycle of the oscillation are largely located in the solvent rather than on the protein or heme, which suggests that the low frequency oscillations in a number of heme proteins, including MbCO, likely originate from a photolysis triggered pressure wave propagating in the solvated protein. Finally, these findings of chemical signals are isolated from coexisting thermal artifacts also by the numerical deconvolution. It is modeled in this study that the ultrashort XFEL pulses cause a transient spike of the local temperature at the heme site of hundreds of K.

Regulation of the Erythrobacter litoralis DSM 8509 general stress response by visible light

Fiebig, A. — 2019-05-19

Extracytoplasmic function (ECF) sigma factors are a major class of environmentally-responsive transcriptional regulators. In Alphaproteobacteria the ECF sigma factor, {sigma}EcfG, activates general stress response (GSR) transcription and protects cells from multiple stressors. A phosphorylation-dependent protein partner switching mechanism, involving HWE/HisKA_2-family histidine kinases, underlies {sigma}EcfG activation. The identity of these sensor kinases and the signals that regulate them remain largely uncharacterized. We have developed the aerobic anoxygenic photoheterotrophic (AAP) bacterium, Erythrobacter litoralis DSM 8509, as a comparative genetic model to investigate GSR regulation. Using this system, we sought to define the contribution of visible light and a photosensory HWE kinase, LovK, to GSR transcription. We identified three HWE kinases that collectively regulate GSR: gsrK and lovK are activators, while gsrP is a repressor. GSR transcription is higher in the dark than light, and the opposing activities of gsrK and gsrP are sufficient to achieve light-dependent differential transcription. In the absence of gsrK and gsrP, lovK alone is sufficient to regulate GSR transcription in response to light. This regulation requires a photochemically active LOV domain in LovK. Our studies establish a role for visible light and HWE kinases in light-dependent regulation of GSR transcription in E. litoralis, an AAP species.nnGRAPHICAL ABSTRACTnnO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/641647v1_ufig1.gif" ALT="Figure 1">nView larger version (19K):norg.highwire.dtl.DTLVardef@65ed9borg.highwire.dtl.DTLVardef@1d25e65org.highwire.dtl.DTLVardef@333ff9org.highwire.dtl.DTLVardef@ad2cf4_HPS_FORMAT_FIGEXP M_FIG C_FIG ABBREVIATED SUMMARYGeneral stress response (GSR) systems protect bacteria from a diverse range of physical and chemical stressors. We have developed Erythrobacter litoralis as a new genetic model to study GSR in Alphaproteobacteria and show that three HWE-family histidine kinases collectively regulate GSR transcription via {sigma}EcfG. Visible light is a GSR regulatory signal in E. litoralis, and LovK is a blue-light photosensor kinase that functions as a dark activated GSR regulator.

The CAFA challenge reports improved protein function prediction and new functional annotations for hundreds of genes through experimental screens

Zhou, N. — 2019-05-29

The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function. Here we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility (P. aureginosa only). We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory. We conclude that, while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. We finally report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bioontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.

Agent-based modeling of persons who inject drugs in metropolitan Chicago suggests that re-treatment with antivirals of persons who are re-infected with Hep C is critical to achieve the WHO incidence reduction objective by 2030

Tatara, E. — 2019-05-31

Background and AimsHepatitis C virus (HCV) infection is a leading cause of chronic liver disease and mortality worldwide. Direct-acting antiviral (DAA) therapy leads to high cure rates. However, persons who inject drugs (PWID) are at risk for reinfection after cure and may require DAA retreatment to reach the World Health Organizations (WHO) goal of HCV elimination by 2030. We aim to project the frequency of retreatment and DAA cost needed to achieve WHO goals. DesignWe use an agent-based model (ABM) that accounts for the complex interplay of demographic factors, risk behaviors, social networks, and geographic location for HCV transmission among PWID. Setting and participants32,000 in-silico PWID in metropolitan Chicago. Intervention and comparatorPossible treatment adherence rates (i.e., DAA cure rates) of 60%-90% with DAA treatment enrollment rates of 2.5%-10% and retreatments per PWID of 0 (retreatment prohibited), 1, 2, 3, or no retreatment restriction were simulated. DAA cost is assumed $25,000 (USD) per treatment. FindingsModeling results indicate that prohibition of retreatment in PWID would jeopardize achieving the WHO goal of reducing the incidence of new chronic HCV infections by 90% by 2030. We predict that with a DAA treatment rate of >7.5% per year and high (90%) adherence, 75%, 19%, 5% and <2% of PWID will require 1, 2, 3, and 4 treatment courses with overall DAA cost of $325 million to achieve the WHO goal in metropolitan Chicago. We estimate a 28% increase in the overall DAA cost under low adherence (70%) compared to high adherence (90%). ConclusionsModeling results predict the frequency of DAA retreatment needed to achieve the WHO goal and underscore the importance of retreatment of HCV re-infections.

Multi-ethnic genome-wide association study of decomposed cardioelectric phenotypes illustrates strategies to identify and characterize evidence of shared genetic effects for complex traits

Baldassari, A. R. — 2019-05-31

BackgroundPublished genome-wide association studies (GWAS) are mainly European-centric, examine a narrow view of phenotypic variation, and infrequently interrogate genetic effects shared across traits. We therefore examined the extent to which a multi-ethnic, combined trait GWAS of phenotypes that map to well-defined biology can enable detection and characterization of complex trait loci.nnMethodsWith 1000 Genomes Phase 3 imputed data in 34,668 participants (15% African American; 3% Chinese American; 51% European American; 30% Hispanic/Latino), we performed covariate-adjusted univariate GWAS of six contiguous electrocardiogram (ECG) traits that decomposed an average heartbeat and two commonly reported composite ECG traits that summed contiguous traits. Combined phenotype testing was performed using the adaptive sum of powered scores test (aSPU).nnResultsWe identified six novel and 87 known ECG trait loci (aSPU p-value < 5E-9). Lead SNP rs3211938 at novel locus CD36 was common in African Americans (minor allele frequency=10%) and near-monomorphic in European Americans, with effect sizes for the composite trait, QT interval, among the largest reported. Only one novel locus was detected for the composite traits, due to opposite directions of effects across contiguous traits that summed to near-zero. Combined phenotype testing did not detect novel loci unapparent by univariate testing. However, this approach aided locus characterization, particularly when loci harbored multiple independent signals that differed by trait.nnConclusionsDespite including one-third as few participants as the largest published GWAS of ECG traits, our study identifies multiple novel ECG genetic loci, emphasizing the importance of ancestral diversity and phenotype measurement in this era of ever-growing GWAS.nnAUTHOR SUMMARYWe leveraged a multiethnic cohort with precise measures of cardioelectric function to identify novel genetic loci affecting this complex, multifaceted phenotype. The success of our approach stresses the importance of phenotypic precision and participant diversity for future locus discovery and characterization efforts, and cautions against compromises made in genome-wide association studies to pursue ever-growing sample sizes.

Assessment of the in vitro function of human stem cell-derived β cells

Srivatsava, A. R. — 2019-05-31

Insulin-producing human embryonic stem cell-derived {beta} (SC-{beta}) cells are a promising cell source for diabetes cell replacement therapy. We have recently reported a differentiation strategy that produces SC-{beta} cells in islet organoids that not only undergo glucose-stimulated insulin secretion but also have an islet-like dynamic insulin release profile, displaying both first and second phase insulin secretion. The goal of this study was to further characterize the functional profile of these SC-{beta} cells in vitro. We utilized a Seahorse extracellular flux analyzer to measure mitochondrial respiration of SC-{beta} cells at low and high glucose. We also used photolithography to fabricate a microfluidic device containing microwells to immobilize SC-{beta} cells for perfusional analysis, monitoring cytoplasmic calcium using Fluo-4 AM at low and high glucose. Here we find that in addition to increased insulin secretion, SC-{beta} cells have increased cellular respiration and cytoplasmic calcium ion concentration in response to a high glucose stimulation. Our results indicate that SC-{beta} cells have similar function to that reported for islets, providing further performance characterization that could help with eventual development for diabetes cell therapy and drug screening.

Genetic, morphometric, and molecular analyses of interspecies differences in head shape and hybrid developmental defects in the wasp genus Nasonia

Cohen, L. B. — 2019-06-07

Males in the parasitoid wasp genus Nasonia (N. vitripennis, N. giraulti, N. longicornis) have distinct, species specific, head shapes. Fertile hybrids among the species are readily produced in the lab allowing genetic analysis of the evolved differences. In addition, the obligate haploidy of males makes these wasps a uniquely powerful model for analyzing the role of complex gene interactions in development and evolution. Previous analyses have shown that complex gene interactions underpin different aspects of the shape differences, and developmental incompatibilities that are specific to the head in F2 haploid hybrid males are also governed by networks of gene interaction. Here we use the genetic tools available in Nasonia to extend our understanding of the gene interactions that affect development and morphogenesis in male heads. Using artificial diploid male hybrids, we show that alleles affecting head shape are codominant, leading to uniform, averaged hybrid F1 diploid male heads, while the alleles mediating developmental defects are recessive, and are not visible in the diploid hybrids. We also determine that divergence in time, rather than in morphological disparity is the primary driver of hybrid developmental defects. In addition, we show that doublesex is necessary for the male head shape differences, but is not the only important factor. Finally we demonstrate that we can dissect complex interspecies gene interaction networks using introgression in this system. These advances represent significant progress in the complex web of gene interactions that govern morphological development, and chart the connections between genomic and phenotypic variation.

Nucleotide-mediated allosteric regulation of bifunctional Rel enzymes

Tamman, H. — 2019-06-13

Bifunctional Rel stringent factors, the most broadly distributed class of RSHs, are ribosome-associated enzymes that transfer a pyrophosphate group from ATP onto the 3' of GTP or GDP to synthesize (p)ppGpp and also catalyse the 3' pyrophosphate hydrolysis of the alarmone to degrade it. The precise regulation of these enzymes seems to be a complex allosteric mechanism, and despite decades of research, it is unclear how the two opposing activities of Rel are controlled at the molecular level. Here we show that a stretch/recoil guanosine-switch mechanism controls the catalytic cycle of T. thermophilus Rel (RelTf). The binding of GDP/ATP stretches apart the NTD catalytic domains of RelTf (RelTtNTD) activating the synthetase domain and allosterically blocking the hydrolase active site. Conversely, binding of ppGpp unlocks the hydrolase domain and triggers recoil of both NTDs, which partially buries the synthetase active site and precludes the binding of synthesis precursors. This allosteric mechanism acts as an activity switch preventing futile cycles of alarmone synthesis and degradation.

Oncoprotein CoAA repeats interact with RNA polymerase II CTD repeats

Xiong, S. — 2019-06-13

The heptad repeating sequence of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II is highly conserved in eukaryotes. In yeast, a CTD code consisting of pairs of heptad repeats is essential for viability. However, the strict requirement of diheptad repeats for the CTD function in transcription and splicing is unexplained. Here we show that CoAA (gene symbol RBM14), an oncoprotein and mammalian transcriptional coactivator, possesses diheptad repeats and directly interacts with the CTD. CoAA comprises 27 copies of tyrosine-rich repeats and regulates pre-mRNA synthesis and alternative splicing. Tyrosine substitutions in either the CoAA repeats or the CTD repeats diminish their interactions. Ser2- or Ser5-phosphorylated CTD peptides exhibit higher binding affinity to CoAA than the corresponding non-phosphorylated CTD peptide. CoAA dynamically interacts with both the CTD and hnRNP M, which is an alternative splicing regulator also comprising diheptad repeats. Arginine methylation of CoAA switches its interaction from the hnRNP M repeats to the CTD repeats. This study provides a mechanism for CoAA at the interface of transcription and alternative splicing, and explains the functional requirement of diheptad repeats in the CTD. In the human genome, tyrosine-rich repeats similar to the CoAA repeats were only found in six oncoproteins including EWS and SYT. We suggest that the diheptad sequence is one of the signature features for the CTD interaction among oncoproteins involved in transcription and alternative splicing. We anticipate that direct RNA Pol II interaction is a mechanism in oncogenesis.

Biofilm inhibitor taurolithocholic acid alters colony morphology, specialized metabolism, and virulence of Pseudomonas aeruginosa

Condren, A. R. — 2019-06-20

Biofilm inhibition by exogenous molecules has been an attractive strategy for the development of novel therapeutics. We investigated the biofilm inhibitor taurolithocholic acid (TLCA) and its effects on the specialized metabolism, virulence and biofilm formation of the clinically relevant bacterium Pseudomonas aeruginosa strain PA14. Our study shows that TLCA alters specialized metabolism, thereby affecting P. aeruginosa colony biofilm physiology. We observed an upregulation of metabolites correlated to virulence such as the siderophore pyochelin. A wax moth virulence assay confirmed that treatment with TLCA increases virulence of P. aeruginosa. Based on our results, we believe that future endeavors to identify biofilm inhibitors must consider how a putative lead is altering the specialized metabolism of a bacterial community to prevent pathogens from entering a highly virulent state.

Correlation between Retinal Ganglion Cell Loss and Nerve Crush Force-Impulse Established with an Instrumented Tweezers in Mice

Liu, X. — 2019-07-01

ObjectivesRodent models of optic nerve crush (ONC) have often been used to study degeneration and regeneration of retinal ganglion cells (RGCs) and their axons as well as the underlying molecular mechanisms. However, ONC results from different laboratories exhibit a range of RGC injury with varying degree of axonal damage. We developed an instrumented tweezers to measure optic nerve (ON) crush forces in real time and studied the correlation between RGC axon loss and force-impulse, the product of force and duration, applied through the instrumented tweezers in mice.nnMethodsA pair of standard self-closing #N7 tweezers were instrumented with miniature foil strain gauges at optimal locations on both tweezer arms. The instrumented tweezers were capable of recording the tip closure forces in the form of voltages, which were calibrated through load cells to corresponding tip closure forces over the operating range. Using the instrumented tweezers, the ONs of multiple mice were crushed with varied forces and durations and the axons in the immunostained sections of the crushed ONs were counted.nnResultsWe found that the surviving axon density correlated with crush force, with longer duration and stronger crush forces producing consistently more axon damage.nnDiscussionThe instrumented tweezers enable a simple technique for measurement of ONC forces in real-time for the first time. Using the instrumented tweezers, experimenters can quantify crush forces during ONC to produce consistent and predictable post-crush cell death. This should permit future studies a way to produce nerve damage more consistently than is available now.

Taxonomically informed scoring enhances confidence in natural products annotation

Rutz, A. — 2019-07-14

Mass spectrometry (MS) hyphenated to liquid chromatography (LC)-MS offers unrivalled sensitivity for metabolite profiling of complex biological matrices encountered in natural products (NP) research. With advanced platforms LC, MS/MS spectra are acquired in an untargeted manner on most detected features. This generates massive and complex sets of spectral data that provide valuable structural information on most analytes. To interpret such datasets, computational methods are mandatory. To this extent, computerized annotation of metabolites links spectral data to candidate structures. When profiling complex extracts spectra are often organized in clusters by similarity via Molecular Networking (MN). A spectral matching score is usually established between the acquired data and experimental or theoretical spectral databases (DB). The process leads to various candidate structures for each MS features. At this stage, obtaining high annotation confidence level remains a challenge notably due to the high chemodiversity of specialized metabolomes.nnThe integration of additional information in a meta-score is a way to capture complementary experimental attributes and improve the annotation process. Here we show that integrating unambiguous taxonomic position of analyzed samples and candidate structures enhances confidence in metabolite annotation. A script is proposed to automatically input such information at various granularity levels (species, genus, and family) and weight the score obtained between experimental spectral data and output of available computational metabolite annotation tools (ISDB-DNP, MS-Finder, Sirius). In all cases, the consideration of the taxonomic distance allowed an efficient re-ranking of the candidate structures leading to a systematic enhancement of the recall and precision rates of the tools (1.5 to 7-fold increase in the F1 score). Our results clearly demonstrate the importance of considering taxonomic information in the process of specialized metabolites annotation. This requires to access structural data systematically documented with biological origin, both for new and previously reported NPs. In this respect, the establishment of an open structural DB of specialized metabolites and their associated metadata (particularly biological sources) is timely and critical for the NP research community.

Phazolicin - a Novel Thiazole/Oxazole-Modified Peptide Inhibiting the Bacterial Ribosome in a Species-Specific Way.

Travin, D. Y. — 2019-07-18

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly expanding and largely untapped class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display an outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium sp. Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants belonging to the Rhizobiales. PHZ inhibits prokaryotic translation through the obstruction of the passage of the nascent peptide through the ribosome exit channel. The cryo-EM structure of the Escherichia coli ribosome with bound PHZ revealed that the drug interacts with the 23S rRNA and ribosomal proteins uL4 and uL22 and obstructs the exit tunnel in a way that is distinct from other compounds blocking the exit channel. We show that the sequence of uL4 ribosomal protein loop involved in PHZ binding determines the species-specificity of antibiotic interaction with its target. PHZ and its predicted homologs from other bacterial species expand the known diversity of LAPs and may be used in the future as biocontrol agents for the needs of agriculture.

A conserved SUMO-Ubiquitin pathway directed by RNF4/SLX5-SLX8 and PIAS4/SIZ1 drives proteasomal degradation of topoisomerase DNA-protein crosslinks

Sun, Y. — 2019-07-19

Topoisomerase cleavage complexes (TOPccs) can be stalled physiologically and by the anticancer drugs camptothecins (TOP1 inhibitors) and etoposide (TOP2 inhibitor), yielding irreversible TOP DNA-protein crosslinks (TOP-DPCs). Here we elucidate how TOP-DPCs are degraded via the SUMO-ubiquitin (Ub) pathway. We show that in human cells, TOP-DPCs are promptly and sequentially conjugated by SUMO-2/3, SUMO-1 and Ub. SUMOylation is catalyzed by the SUMO ligase PIAS4, which forms a complex with both TOP1 and TOP2 and {beta}. RNF4 acts as the SUMO-targeted ubiquitin ligase (STUbL) for both TOP1- and TOP2-DPCs in a SUMO-dependent but replication/transcription-independent manner. This SUMO-Ub pathway is conserved in yeast with Siz1 the ortholog of PIAS4 and Slx5-Slx8 the ortholog of RNF4. Our study reveals a conserved SUMO-dependent ubiquitylation pathway for proteasomal degradation of both TOP1- and TOP2-DPCs and potentially for other DPCs.nnnnO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/707661v1_ufig1.gif" ALT="Figure 1">nView larger version (26K):norg.highwire.dtl.DTLVardef@b00b16org.highwire.dtl.DTLVardef@1a81254org.highwire.dtl.DTLVardef@14b75c1org.highwire.dtl.DTLVardef@12000ba_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefTopoisomerase DNA-protein crosslinks (TOP-DPCs) are the therapeutic mechanism of clinical TOP inhibitors (camptothecin and etoposide). TOP-DPCs induce rapid and sequential conjugation of SUMO-2/3- SUMO-1 and ubiquitin catalyzed by activation of PIAS4 through its DNA-binding SAP domain and RNF4 through its SIM domains. This SUMO-ubiquitin cascade triggers proteasomal degradation of TOP-DPCs.nnHIGHLIGHTSO_LIAbortive topoisomerase I (TOP1) and II (TOP2) cleavage complexes resulting in DNA-protein crosslinks (TOP-DPCs) are rapidly and sequentially modified by SUMO-2/3, SUMO-1 and ubiquitin before their proteasomal degradation.nC_LIO_LIPIAS4 SUMOylates TOP-DPCs via its DNA-binding SAP domain independently of DNA transactions and DNA damage responses.nC_LIO_LIRNF4 ubiquitylates SUMOylated TOP-DPCs and drives their proteasomal degradation.nC_LIO_LITOP-DPC processing by the SUMO-Ub pathways is conserved in yeast and human cells.nC_LI

In-silico definition of the Drosophila melanogaster matrisome

Davis, M. N. — 2019-08-02

The extracellular matrix (ECM) is an assembly of hundreds of proteins that structurally supports the cells it surrounds and biochemically regulates their functions. Drosophila has emerged as a powerful model organism to study fundamental mechanisms underlying ECM protein secretion, ECM assembly, and ECM roles in pathophysiological processes. However, as of today, we do not possess a well-defined list of the components forming the ECM of this organism. We previously reported the development of computational pipelines to define the matrisome - the ensemble of genes encoding ECM and ECM-associated proteins - of humans, mice, zebrafish and C. elegans. Using a similar approach, we report here that the Drosophila matrisome is composed of 641 genes. We further classify these genes into different structural and functional categories, including an expanded way to classify genes encoding proteins forming apical ECMs. We illustrate how having a comprehensive list of Drosophila matrisome proteins can be used to annotate large proteomic datasets and identify unsuspected roles for the ECM in pathophysiological processes. Last, to aid the dissemination and usage of the proposed definition and categorization of the Drosophila matrisome by the scientific community, our list has been made available through three public portals: The Matrisome Project, FlyBase, and GLAD.

Release of stem cells from quiescence reveals multiple gliogenic domains in the adult brain

Delgado, A. — 2019-08-16

Quiescent neural stem cells (NSCs) in the adult ventricular-subventricular zone (V-SVZ) have a regional identity and undergo activation to generate neurons. The domains for gliogenesis are less explored. Here we show that Platelet-Derived Growth Factor Receptor beta (PDGFR{beta}) is expressed by adult V-SVZ NSCs that generate olfactory bulb interneurons and glia with slow baseline kinetics. Selective deletion of PDGFR{beta} in adult V-SVZ NSCs leads to their release from quiescence uncovering multiple domains in the septal wall for oligodendrocyte and astrocyte formation. Unexpectedly, we identify a novel intraventricular oligodendrocyte progenitor inside the brain ventricles. Together our findings reveal different NSC spatial domains for gliogenesis in the adult V-SVZ that are largely quiescent under homeostasis and may have key functions for brain plasticity.

Disruption of NOX2-dependent Oxidative Injury with a Targeted Gene-Therapy Approach Prevents Atrial Fibrillation in a Canine Model

Yoo, S. — 2019-09-11

Atrial fibrillation is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments of AF are suboptimal as they are not targeted to the molecular mechanisms underlying AF. In this study, we demonstrated using a novel gene-based strategy in a clinically relevant large animal of AF that oxidative injury is a key mechanism underlying the onset and perpetuation of AF. First, we demonstrated that generation of oxidative injury in atrial myocytes is a frequency-dependent process, with rapid pacing in canine atrial myocytes inducing oxidative injury through induction of NADPH oxidase 2 (NOX2) and generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electrical remodeling in AF by upregulating a constitutively active form of acetylcholine-dependent K+ current (IKACh) - called IKH - by a mechanism involving frequency-dependent activation of protein kinase C epsilon (PKC{varepsilon}). To understand the mechanism by which oxidative injury promotes the genesis and/or maintenance of AF, we performed targeted injection of NOX2 shRNA in atria of normal dogs followed by rapid atrial pacing. The time to onset of non-sustained AF increased by more than 5-fold in NOX2 shRNA treated dogs. Furthermore, animals treated with NOX2 shRNA did not develop sustained AF for up to 12 weeks. The electrophysiological mechanism underlying AF prevention was prolongation of atrial effective refractory periods, with attenuated activation of PKC{varepsilon}, a likely molecular mechanism underlying this beneficial electrophysiological remodeling. Future optimization of this approach may lead to a novel, mechanism-guided therapy for AF.nnOne Sentence SummaryTargeted disruption of NOX2-dependent oxidative injury with a novel gene therapy approach prevents onset as well as perpetuation of atrial fibrillation.

DMSO potentiates the suppressive effect of dronabinol, a cannabinoid, on sleep apnea and REM sleep

Calik, M. W. — 2019-09-18

PurposeDimethyl sulfoxide (DMSO) is an amphipathic molecule with innate biological activity that also is used to dissolve both polar and nonpolar compounds in preclinical and clinical studies. Recent investigations of dronabinol, a cannabinoid, dissolved in DMSO demonstrated decreased sleep apnea frequency and time spent in REM sleep in rats. Here, we tested the effects of dronabinol dissolved in 25% DMSO (diluted in phosphate-buffered saline) to rule out potentiating effects of DMSO.nnMethodsSprague-Dawley rats were anesthetized and implanted with bilateral stainless steel screws into the skull for electroencephalogram recording and bilateral wire electrodes into the nuchal muscles for electromyogram recording. Each animal was recorded by polysomnography. The study was a fully nested, repeated measures crossover design, such that each rat was recorded following each of 8 intraperitoneal injections separated by three days: vehicle (25% DMSO/PBS); vehicle and CB1 antagonist (AM 251); vehicle and CB2 antagonist (AM 630); vehicle and CB1/CB2 antagonist; dronabinol (CB1/CB2 agonist); dronabinol and CB1 antagonist; dronabinol and CB2 antagonist; and dronabinol and CB1/CB2 antagonist. Sleep was manually scored into NREM and REM stages, and apneas were quantified.nnResultsDronabinol dissolved in 25% DMSO did not suppress apnea or modify sleep efficiency compared to vehicle controls, in contrast to previously published results. However, dronabinol did suppress REM sleep, which is in line with previously published results.nnConclusionsDronabinol in 25% DMSO partially potentiated dronabinols effects, suggesting a concomitant biological effect of DMSO on breathing during sleep.

Inherited Causes of Clonal Hematopoiesis of Indeterminate Potential in TOPMed Whole Genomes

Bick, A. G. — 2019-09-27

Age is the dominant risk factor for most chronic human diseases; yet the mechanisms by which aging confers this risk are largely unknown.1 Recently, the age-related acquisition of somatic mutations in regenerating hematopoietic stem cell populations was associated with both hematologic cancer incidence2-4 and coronary heart disease prevalence.5 Somatic mutations with leukemogenic potential may confer selective cellular advantages leading to clonal expansion, a phenomenon termed Clonal Hematopoiesis of Indeterminate Potential (CHIP).6 Simultaneous germline and somatic whole genome sequence analysis now provides the opportunity to identify root causes of CHIP. Here, we analyze high-coverage whole genome sequences from 97,691 participants of diverse ancestries in the NHLBI TOPMed program and identify 4,229 individuals with CHIP. We identify associations with blood cell, lipid, and inflammatory traits specific to different CHIP genes. Association of a genome-wide set of germline genetic variants identified three genetic loci associated with CHIP status, including one locus at TET2 that was African ancestry specific. In silico-informed in vitro evaluation of the TET2 germline locus identified a causal variant that disrupts a TET2 distal enhancer. Aggregates of rare germline loss-of-function variants in CHEK2, a DNA damage repair gene, predisposed to CHIP acquisition. Overall, we observe that germline genetic variation altering hematopoietic stem cell function and the fidelity of DNA-damage repair increase the likelihood of somatic mutations leading to CHIP.

Hedgehog pathway activation through conformational blockade of the Patched sterol conduit

Zhang, Y. — 2019-09-26

Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental and therapeutic application has been difficult for lack of agents amenable to tissue targeting. We have developed a novel agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1. This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 "switch helix", as revealed by cryo-EM structure determination. Although this conformation likely constitutes part of the transport cycle, nanobody-trapping disrupts the cycle and prevents substrate movement through the Patched1 sterol conduit. Our conformation-selective nanobody approach provides a new route to the development of transporter-related pharmacologic agents and may be generally applicable to the study of other transporters.

Scaffold-free human mesenchymal stem cell construct geometry regulates long bone regeneration

Herberg, S. — 2019-09-30

Scaffold-based bone tissue engineering approaches frequently induce repair processes dissimilar to normal developmental programs. In contrast, biomimetic strategies aim to recapitulate aspects of development through cellular self-organization, morphogenetic pathway activation, and mechanical cues. This may improve regenerative outcome in large long bone defects that cannot heal on their own; however, no study to date has investigated the role of scaffold-free construct geometry, in this case tubes mimicking long bone diaphyses, on bone regeneration. We hypothesized that microparticle-mediated in situ presentation of transforming growth factor-{beta}1 (TGF-{beta}1) and bone morphogenetic protein-2 (BMP-2) to engineered human mesenchymal stem cell (hMSC) tubes induces the endochondral cascade, and that TGF-{beta}1 + BMP-2-presenting hMSC tubes facilitate enhanced endochondral healing of critical-sized femoral segmental defects under delayed in vivo mechanical loading conditions compared to loosely-packed hMSC sheets. Here, localized morphogen presentation imparted early chondrogenic lineage priming, and stimulated robust endochondral differentiation of hMSC tubes in vitro. In an ectopic environment, hMSC tubes formed a cartilage template that was actively remodeled into trabecular bone through endochondral ossification without lengthy predifferentiation. Similarly, hMSC tubes stimulated in vivo cartilage and bone formation and more robust healing in femoral defects compared to hMSC sheets. New bone was formed through endochondral ossification in both groups; however, only hMSC tubes induced regenerate tissue partially resembling normal growth plate architecture. Together, this study demonstrates the interaction between mesenchymal cell condensation geometry, bioavailability of multiple morphogens, and defined in vivo mechanical environment to recapitulate developmental programs for biomimetic bone tissue engineering.nnSignificance StatementEngineered bone constructs must be capable of withstanding and adapting to harsh conditions in a defect site upon implantation, and can be designed to facilitate repair processes that resemble normal developmental programs. Self-assembled tubular human mesenchymal stem cell constructs were engineered to resemble the geometry of long bone diaphyses. By mimicking the cellular, biochemical, and mechanical environment of the endochondral ossification process during embryonic development, successful healing of large femoral segmental defects upon implantation was achieved and the extent was construct geometry dependent. Importantly, results were obtained without a supporting scaffold or lengthy predifferentiation of the tubular constructs. This indicates that adult stem/progenitor cells retain features of embryonic mesenchyme, and supports the concept of developmental engineering for bone regeneration approaches.

TAK1 Regulates Endothelial Integrity Through Stabilization of Junctions via GSK3β and FoxO1

Regmi, S. C. — 2019-09-27

TLR4 signaling in endothelial cells (ECs) induces vascular injury by disrupting the endothelial junctional barrier. However, it is not known whether TLR4 signaling can also promote endothelial barrier repair after vascular injury. Here we addressed the role of TAK1 activation downstream of TLR4 in the mechanism of vascular integrity. In inducible EC-restricted TAK1 knockout (TAK1i{Delta}EC) mice, the endothelial barrier was compromised. Blocking TAK1 activity caused spontaneous loss of the endothelial barrier. Importantly, TAK1 inactivated GSK3{beta} via AKT to prevent {beta}-catenin downregulation. We observed in ECs of GSK3{beta}i{Delta}EC mice an increase in {beta}-catenin transfer to the nucleus to form a complex with transcription factor FoxO1, thus repressing the expression of the tight junction protein claudin-5 and causing vascular leak. Strikingly, in TAK1i{Delta}EC mice, FoxO1 expression was dramatically increased while expression of AKT was suppressed, and in vivo inhibition of FoxO1 prevented sepsis-induced lung vascular leak in GSK3{beta}i{Delta}EC and TAK1i{Delta}EC mice. Further, EC-restricted deletion of FoxO1 in mice suppressed sepsis-induced lung vascular leak and mortality. Our findings point to the potential of targeting the TAK1-AKT-GSK3{beta}-FoxO1 axis as a therapeutic approach to treat uncontrolled lung vascular leak in sepsis.

α-ketoglutaric acid stimulates muscle hypertrophy and fat loss through OXGR1-dependent adrenal activation

Shu, G. — 2019-11-05

Beneficial effects of resistance exercise on metabolic health and particularly muscle hypertrophy and fat loss are well established, but the underlying chemical and physiological mechanisms are not fully understood. Here we identified a myometabolite-mediated metabolic pathway that is essential for the beneficial metabolic effects of resistance exercise in vivo. We showed that substantial accumulation of the tricarboxylic acid cycle intermediate -ketoglutaric acid (AKG) is a metabolic signature of resistance exercise performance. Interestingly, human plasma AKG level is also negatively correlated with BMI. Pharmacological elevation of circulating AKG induces muscle hypertrophy, brown adipose tissue (BAT) thermogenesis, and white adipose tissue (WAT) lipolysis in vivo. We further found that AKG stimulates the adrenal release of adrenaline through 2-oxoglutarate receptor 1 (OXGR1) expressed in adrenal glands. Finally, by using both loss-of-function and gain-of-function mouse models, we showed that OXGR1 is essential for AKG-mediated exercise-induced beneficial metabolic effects. These findings reveal an unappreciated mechanism for the salutary effects of resistance exercise, using AKG as a systemically-derived molecule for adrenal stimulation of muscle hypertrophy and fat loss.

Estrogen Receptor α Regulates Ethanol Excitation of Ventral Tegmental Area Neurons and Binge Drinking in Female Mice

Vandegrift, B. J. — 2019-10-10

Elevations in estrogen (17{beta}-estradiol, E2) are associated with increased alcohol drinking by women and experimentally in rodents. E2 alters the activity of the dopamine system, including the ventral tegmental area (VTA) and its projection targets, which plays an important role in binge drinking. A previous study demonstrated that during high E2 states, VTA dopamine neurons in female mice are more sensitive to ethanol excitation. However, the mechanisms responsible for the ability of E2 to enhance ethanol sensitivity of VTA dopamine neurons have not been investigated. In this study, we used selective agonists and antagonists to examine the role of estrogen receptor subtypes (ER and ER{beta}) in regulating the ethanol sensitivity of VTA dopamine neurons and found that ER promotes the enhanced ethanol response of VTA dopamine neurons. We also demonstrated that the E2-induced increase in ethanol excitation requires the activity of the metabotropic glutamate receptor, mGluR1, which is known to couple with ER at the plasma membrane. To investigate the behavioral relevance of these findings, we administered lentivirus expressing short hairpin RNAs targeting either ER or ER{beta} into the VTA and found that knockdown of each receptor in the VTA reduced binge-like ethanol drinking in female, but not male, mice. Reducing ER in the VTA had a more dramatic effect on binge-like drinking than reducing ER{beta}, consistent with the ability of ER to alter ethanol sensitivity of dopamine neurons. These results provide important insight into sex-specific mechanisms that drive excessive alcohol drinking.nnSignificance StatementEstrogen has potent effects on the dopamine system and increases the vulnerability of females to develop addiction to substances such as cocaine and alcohol. We investigated the mechanisms by which estrogen increases the response of dopamine neurons in the ventral tegmental area to ethanol. We found that activation of the estrogen receptor, ER, increased the ethanol-induced excitation of dopamine neurons and that this required the metabotropic glutamate receptor mGluR1. We also demonstrated that estrogen receptors in the ventral tegmental area regulate binge-like alcohol drinking by female, but not male, mice. The influence of estrogen receptors on binge drinking in female mice suggests that treatments for alcohol use disorder in women may need to account for this sex difference.

Paralytic, the Drosophila voltage-gated sodium channel, regulates proliferation of neural progenitors

Piggott, B. J. — 2019-10-14

Proliferating cells, typically considered "non-excitable," nevertheless exhibit regulation by bioelectrical signals. Notably, voltage-gated sodium channels (VGSC) that are crucial for neuronal excitability, are also found in progenitors and upregulated in cancer. Here, we identify a role for VGSC in proliferation of Drosophila neuroblast (NB) lineages within the central nervous system. Loss of paralytic (para), the sole gene that encodes Drosophila VGSC, reduces neuroblast progeny cell number. The type II neuroblast lineages, featuring a transit-amplifying intermediate neural progenitors (INP) population similar to that found in the developing human cortex, are particularly sensitive to para manipulation. Following a series of asymmetric divisions, INPs normally exit the cell cycle through a final symmetric division. Our data suggests that loss of para induces apoptosis in this population, whereas overexpression leads to an increase in INPs and overall neuroblast progeny cell numbers. These effects are cell autonomous and depend on Para channel activity. Reduction of Para not only affects normal NB development, but also strongly suppresses brain tumor mass, implicating a role for Para in cancer progression. To our knowledge, our studies are the first to identify a role for VGSC in neural progenitor proliferation. Elucidating the contribution of VGSC in proliferation will advance our understanding of bioelectric signaling within development and disease states.

A single-cell transcriptome atlas of the adult muscle precursors uncovers early events in fiber-type divergence in Drosophila

Zappia, M. P. — 2019-10-16

In Drosophila, the wing disc-associated adult muscle precursors (AMPs) give rise to the fibrillar indirect flight muscles (IFM) and the tubular direct flight muscles (DFM). To understand early transcriptional events underlying this muscle diversification, we performed single cell RNA-sequencing experiments and built a cell atlas of AMPs associated with third instar larval wing disc. Our analysis identified distinct transcriptional signatures for IFM and DFM precursors that underlie the molecular basis of their divergence. The atlas further revealed various states of differentiation of AMPs, thus illustrating previously unappreciated spatial and temporal heterogeneity among them. We identified and validated novel markers for both IFM and DFM precursors at various states of differentiation by immunofluorescence and genetic tracing experiments. Finally, we performed a systematic genetic screen using a panel of markers from the reference cell atlas as an entry point and found a novel gene, Ama, which is functionally important in muscle development. Thus, our work provides a framework of leveraging scRNA-seq for gene discovery and therefore, this strategy can be applied to other scRNA-seq datasets.

Phenylpropionc acid produced by gut microbiota alleviates acetaminophen-induced hepatotoxicity

Cho, S. — 2019-10-21

ABSTRACTAcetaminophen (APAP) overdose causes hepatic injury and is major contributor to acute liver injury cases. To investigate potential roles of gut microbiota in APAP-induced liver injury, C57BL/6 mice from Jackson (JAX) or Taconic (TAC) were challenged with APAP. TAC mice were more susceptible to APAP toxicity, and this disappeared upon co-housing of JAX and TAC mice. When the cecum contents from JAX and TAC mice were transplanted to germ-free mice, the mice that received TAC gut microbiota exhibited more significant hepatotoxicity after APAP administration. Non-targeted metabolomic analysis using portal vein serum and liver tissue of the mice led to identification of 19 metabolites the levels of which are associated with JAX or TAC gut microbiota. A gut bacteria-derived metabolite phenylpropionic acid (PPA) levels in cecum contents and blood were higher in mice harboring JAX gut microbiota. PPA supplementation in drinking water alleviated APAP-induced hepatotoxicity in TAC mice. This was accompanied by reduced hepatic protein levels of cytochrome P450 (CYP) 2E1, the enzyme responsible for APAP bioactivation to a toxic metabolite. This illustrates a gut microbe-liver interaction mediated by a gut bacteria-derived metabolite in modulating drug-induced liver injury.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Intermittent Starvation Extends the Functional Lifetime of Primary Human Hepatocyte Cultures

Davidson, M. — 2019-10-25

Primary human hepatocyte (PHH) cultures have become indispensable to mitigate the risk of adverse drug reactions in human patients. In contrast to de-differentiating monocultures, co-culture with non-parenchymal cells maintains PHH functions for 2-4 weeks. However, since the functional lifespan of PHHs in vivo is 200-400 days, it is desirable to further prolong PHH functions in vitro towards modeling chronic drug exposure and disease progression. Fasting has benefits on the longevity of organisms and the health of tissues such as the liver. We hypothesized that a culturing protocol that mimics dynamic fasting/starvation could activate starvation pathways and prolong PHH functional lifetime. To mimic starvation, serum and hormones were intermittently removed from the culture medium of micropatterned co-cultures (MPCC) containing PHHs organized onto collagen domains and surrounded by 3T3-J2 murine fibroblasts. A weekly 2-day starvation optimally prolonged PHH functional lifetime for 6+ weeks in MPCCs versus a decline after 3 weeks in non-starved controls. The 2-day starvation also enhanced the functions of PHH monocultures for 2 weeks, suggesting direct effects on PHHs. In MPCCs, starvation activated adenosine monophosphate activated protein kinase and restricted fibroblast overgrowth onto PHH islands, thereby maintaining hepatic polarity. The effects of starvation on MPCCs were partially recapitulated by activating adenosine monophosphate activated protein kinase using metformin or growth-arresting fibroblasts via mitomycin-C. Lastly, starved MPCCs demonstrated lower false positives for drug toxicity tests and higher drug-induced cytochrome-P450 activities versus non-starved controls even after 5 weeks. In conclusion, intermittent serum/hormone starvation extends PHH functional lifetime towards enabling clinically-relevant drug screening.

A novel dynamic network imaging analysis method reveals aging-related fragmentation of cortical networks in mouse

Llano, D. A. — 2019-11-09

Network analysis of large-scale neuroimaging data has proven to be a particularly challenging computational problem. In this study, we adapt a novel analytical tool, known as the community dynamic inference method (CommDy), which was inspired by social network theory, for the study of brain imaging data from an aging mouse model. CommDy has been successfully used in other domains in biology; this report represents its first use in neuroscience. We used CommDy to investigate aging-related changes in network parameters in the auditory and motor cortices using flavoprotein autofluorescence imaging in brain slices and in vivo. Analysis of spontaneous activations in the auditory cortex of slices taken from young and aged animals demonstrated that cortical networks in aged brains were highly fragmented compared to networks observed in young animals. Specifically, the degree of connectivity of each activated node in the aged brains was significantly lower than those seen in the young brain, and multivariate analyses of all derived network metrics showed distinct clusters of these metrics in young vs. aged brains. CommDy network metrics were then used to build a random-forests classifier based on NMDA-receptor blockade data, which successfully recapitulated the aging findings, suggesting that the excitatory synaptic substructure of the auditory cortex may be altered during aging. A similar aging-related decline in network connectivity was also observed in spontaneous activity obtained from the awake motor cortex, suggesting that the findings in the auditory cortex are reflections of general mechanisms that occur during aging. Therefore, CommDy therefore provides a new dynamic network analytical tool to study the brain and provides links between network-level and synaptic-level dysfunction in the aging brain.

Addition of insoluble fiber to isolation media allows for increased metabolite diversity of lab-cultivable microbes derived from zebrafish gut samples

Condren, A. R. — 2019-11-25

There is a gap in measured microbial diversity when comparing genomic sequencing techniques versus cultivation from environmental samples in a laboratory setting. Standardized methods in artificial environments may not recapitulate the environmental conditions that native microbes require for optimal growth. For example, the intestinal tract houses microbes at various pH values as well as minimal oxygen and light environments. These microbes are also exposed to an atypical source of carbon: dietary fiber compacted in fecal matter. To investigate how the addition of insoluble fiber to isolation media could affect the cultivation of microbes from zebrafish intestines, an isolate library was built and analyzed using the bioinformatics pipeline IDBac. The addition of fiber led to an increase in bacterial growth and encouraged the growth of species from several phyla. Furthermore, fiber addition altered the metabolism of the cultivated gut-derived microbes and induced the production of unique metabolites that were not produced when microbes were otherwise grown on standard isolation media. Addition of this inexpensive carbon source to media supported the cultivation of a diverse community whose specialized metabolite production may more closely replicate their metabolite production in vivo.

CHROMATIX: computing the functional landscape of many-body chromatin interactions in transcriptionally active loci

Perez-Rathke, A. — 2019-11-25

Chromatin interactions are important for gene regulation and cellular specialization. Emerging evidence suggests many-body spatial interactions can play important roles in condensing super-enhancer regions into a cohesive transcriptional apparatus. Chromosome conformation studies using Hi-C are limited to pairwise, population-averaged interactions; therefore, not suitable for direct assessment of many-body interactions. We describe a computational model, CHROMATIX, that reconstructs structural ensembles based on Hi-C data and identifies significant many-body interactions. For a diverse set of highly-active transcriptional loci with at least 2 super-enhancers, we detail the many-body functional landscape and show DNase-accessibility, POLR2A binding, and decreased H3K27me3 are predictive of interaction-enriched regions.

High-Resolution Single-Cell Models of Ensemble Chromatin Structures during Drosophila Embryogenesis from Population Hi-C

Sun, Q. — 2019-11-25

Single-cell chromatin studies provide insights into how chromatin structure relates to functions of individual cells. However, balancing high-resolution and genome wide-coverage remains challenging. We describe a computational method for the reconstruction of large 3D-ensembles of single-cell (sc) chromatin conformations from population Hi-C that we apply to study embryogenesis in Drosophila. With minimal assumptions of physical properties and without adjustable parameters, our method generates large ensembles of chromatin conformations via deep-sampling. Our method identifies specific interactions, which constitute 5-6% of Hi-C frequencies, but surprisingly are sufficient to drive chromatin folding, giving rise to the observed Hi-C patterns. Modeled sc-chromatins quantify chromatin heterogeneity, revealing significant changes during embryogenesis. Furthermore, >50% of modeled sc-chromatin maintain topologically associating domains (TADs) in early embryos, when no population TADs are perceptible. Domain boundaries become fixated during development, with strong preference at binding-sites of insulator-complexes upon the mid-blastula transition. Overall, high-resolution 3D-ensembles of sc-chromatin conformations enable further in-depth interpretation of population Hi-C, improving understanding of the structure-function relationship of genome organization.

Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in 3-Dimensional Models

Kukla, D. — 2019-11-29

Human liver models that are 3-dimensional (3D) in architecture are proving to be indispensable for diverse applications, including compound metabolism and toxicity screening during preclinical drug development, to model human liver diseases for the discovery of novel therapeutics, and for cell-based therapies in the clinic; however, further development of such models is needed to maintain high levels of primary human hepatocyte (PHH) functions for weeks to months in vitro. Therefore, here we determined how microscale 3D collagen-I presentation and fibroblast interaction could affect the long-term functions of PHHs. High-throughput droplet microfluidics was utilized to rapidly generate reproducibly-sized (~300 m diameter) microtissues containing PHHs encapsulated in collagen-I +/- supportive fibroblasts, namely 3T3-J2 murine embryonic fibroblasts or primary human hepatic stellate cells (HSCs); self-assembled spheroids and bulk collagen gels (macrogels) containing PHHs served as gold-standard controls. Hepatic functions (e.g. albumin and cytochrome-P450 or CYP activities) and gene expression were subsequently measured for up to 6 weeks. We found that collagen-based 3D microtissues rescued PHH functions within static multi-well plates at 2- to 30-fold higher levels than self-assembled spheroids or macrogels. Further coating of PHH microtissues with 3T3-J2s led to higher hepatic functions than when the two cell types were either coencapsulated together or when HSCs were used for the coating instead. Additionally, the 3T3-J2-coated PHH microtissues displayed 6+ weeks of relatively stable hepatic gene expression and function at levels similar to freshly thawed PHHs. Lastly, microtissues responded in a clinically-relevant manner to drug-mediated CYP induction or hepatotoxicity. In conclusion, fibroblast-coated collagen microtissues containing PHHs display hepatic functions for 6+ weeks without any fluid perfusion at higher levels than spheroids and macrogels, and such microtissues can be used to assess drug-mediated CYP induction and hepatotoxicity. Ultimately, microtissues may find broader utility for modeling liver diseases and as building blocks for cell-based therapies.

Highly clustered de novo frameshift variants in the neuronal splicing factor NOVA2 result in a specific abnormal C terminal part and cause a severe form of intellectual disability with autistic features.

Mattioli, F. — 2019-12-04

The Neuro-Oncological Ventral Antigen 2 NOVA2 protein is a major factor regulating neuron specific alternative splicing, previously associated with an acquired neurologic condition, the paraneoplastic opsoclonus-myoclonus ataxia (POMA). We report here six individuals with de novo frameshift variants in the NOVA2 gene affected with a severe neurodevelopmental disorder characterized by intellectual disability (ID), motor and speech delay, autistic features, hypotonia, feeding difficulties, spasticity or ataxic gait and abnormal brain MRI. The six variants lead to the same reading frame, adding a common 133 aa long proline rich C-terminus part instead of the last KH RNA binding domain. We detected forty-one genes differentially spliced after NOVA2 inactivation in human neural cells. The mutant NOVA2 protein shows decreased ability to bind a target RNA, to regulate specific splicing events and to rescue the phenotype of altered retinotectal axonal pathfinding induced by loss of NOVA2 ortholog in zebrafish. Our results suggest a partial loss-of-function mechanism rather than a full heterozygous loss of function, although a specific contribution of the novel C terminal extension cannot be excluded on the basis of the genetic findings.

Multiplex Imaging Reveals Novel Subcellular, Microenvironmental, and Racial Patterns of MRTFA/B Activation in Invasive Breast Cancers and Metastases

Wilk, S. M. — 2024-01-08

Breast cancer progression and metastasis involve the action of multiple transcription factors in tumors and in the cells of the tumor microenvironment (TME) and understanding how these transcription factors are coordinated can guide novel therapeutic strategies. Myocardin related transcription factors A and B (MRTFA/B) are two related transcription factors that redundantly control cancer cell invasion and metastasis in mouse models of breast cancer, but their roles in human cancer are incompletely understood. Here, we used a combination of multiplexed immunofluorescence and bioinformatics analyses to show that MRTFA/B are concurrently activated in tumor cells, but they show distinct patterns of expression across different histological subtypes and in the TME. Importantly, MRTFA expression was elevated in metastatic tumors of African American patients, who disproportionately die from breast cancer. Interestingly, in contrast to publicly available mRNA expression data, MRTFA was similarly expressed across estrogen receptor (ER) positive and negative breast tumors, while MRTFB expression was highest in ER+ breast tumors. Furthermore, MRTFA was specifically expressed in the perivascular antigen presenting cells (APCs) and its expression correlated with the expression of the immune checkpoint protein V-set immunoregulatory receptor (VSIR). These results provide unique insights into how MRTFA and MRTFB can promote metastasis in human cancer, into the racial disparities of their expression patterns, and their function within the complex breast cancer TME. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/573909v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@12037b3org.highwire.dtl.DTLVardef@28e2feorg.highwire.dtl.DTLVardef@4e95e1org.highwire.dtl.DTLVardef@152019b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Towards Real-Time Airborne Pathogen Sensing: Electrostatic Capture and On-Chip LAMP Based Detection of Airborne Viral Pathogens

Jaykumar, N. S. — 2024-01-08

Considerable loss of life, economic slowdown, and public health risk associated with the transmission of airborne respiratory pathogens was underscored by the recent COVID-19 pandemic. Airborne transmission of zoonotic diseases such as the highly pathogenic avian influenza (HPAI) and porcine reproductive and respiratory syndrome virus (PRRSV) has caused major disruptions to domestic and global food security. Current ambient air pathogen monitoring systems involves the collection of air samples from indoor settings suspected of viral contamination, followed by subsequent processing of capture samples to determine the presence and species of airborne viral matter. Nucleic acid amplification techniques are considered the gold standard for pathogen diagnostics. Currently, the necessary extraction and purification of viral RNA from air collector systems prior to sample analysis is both time consuming and performed manually. A monitoring system with separate air sampling and biochemical detection procedures is prone to delay the response to emergent viral threats. In this paper, we present a pathogen monitoring system that overcomes these limitations related to extraction and purification of viral samples and lays the groundwork for a real-time monitor for airborne viral pathogens. We demonstrate a high flow electrostatic precipitator system, that uses small collection wells as counter electrodes for pathogen collection. Integrated reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) is used for detection of captured viral matter within wells. On-chip heating of collection wells is enabled by integrated planar heaters and small volumes of reagent (30 L) directly to the collection wells. We present the design of such a system and show experimental results that demonstrate the use of this device for detection of aerosolized SARS-CoV-2 virus like particles (VLPs), a model pathogen for SARV-CoV-2.

Epitranscriptomic Reader YTHDF2 Regulates SEK1(MAP2K4)-JNK-cJUN Inflammatory Signaling in Astrocytes during Neurotoxic Stress

Malovic, E. — 2024-01-26

As the most abundant glial cells in the CNS, astrocytes dynamically respond to neurotoxic stress, however, the key molecular regulators controlling the inflammatory status of these sentinels during neurotoxic stress have remained elusive. Herein, we demonstrate that the m6A epitranscriptomic mRNA modification tightly regulates the pro-inflammatory functions of astrocytes. Specifically, the astrocytic neurotoxic stresser, manganese (Mn), downregulated the m6A reader YTHDF2 in human and mouse astrocyte cultures and in the mouse brain. Functionally, YTHDF2 knockdown augmented, while its overexpression dampened, neurotoxic stress induced proinflammatory response, suggesting YTHDF2 serves as a key upstream regulator of inflammatory responses in astrocytes. Mechnistically, YTHDF2 RIP-sequencing identified MAP2K4 (MKK4; SEK1) mRNA as a YTHDF2 target influencing inflammatory signaling. Our target validation revealed Mn-exposed astrocytes mediates proinflammatory response by activating the phosphorylation of SEK1, JNK, and cJUN signaling. Collectively, YTHDF2 serves a key upstream molecular switch controlling SEK1(MAP2K4)-JNK-cJUN proinflammatory signaling in astrocytes.

Temporal Dynamics of Cyanobacterial Bloom Community Composition and Toxin Production from Urban Lakes

Maurer, J. A. — 2024-02-10

With a long evolutionary history and a need to adapt to a changing environment, cyanobacteria in freshwater systems use specialized metabolites for communication, defense, and physiological processes. However, the role that these metabolites play in differentiating species, maintaining microbial communities, and generating niche persistence and expansion is poorly understood. Furthermore, many cyanobacterial specialized metabolites and toxins present significant human health concerns due to their liver toxicity and their potential impact to drinking water. Gaps in knowledge exist with respect to changes in species diversity and toxin production during a cyanobacterial bloom (cyanoHAB) event; addressing these gaps will improve understanding of impacts to public and ecological health. In the current project, we utilized a multi-omics strategy (DNA metabarcoding and metabolomics) to determine the cyanobacterial community composition, toxin profile, and the specialized metabolite pool at three freshwater lakes in Providence, RI during summer-fall cyanoHABs. Species diversity decreased at all study sites over the course of the bloom event, and toxin production reached a maximum at the midpoint of the event. Additionally, LC-MS/MS-based molecular networking identified new toxin congeners. This work provokes intriguing questions with respect to the use of allelopathy by organisms in these systems and the presence of emerging toxic compounds that can impact public health. SYNOPSISThis study reports on cyanobacterial community succession and toxin dynamics during cyanobacterial bloom events. Results show relationships and temporal dynamics that are relevant to public health.

Genetic polymorphisms of Leukocyte Immunoglobulin-Like Receptor B3 (LILRB3) gene in African American kidney transplant recipients are associated with post-transplant graft failure

Sun, Z. — 2024-02-23

BackgroundAfrican American (AA) kidney transplant recipients exhibit a higher rate of graft loss compared to other racial and ethnic populations, highlighting the need to identify causative factors underlying this disparity. MethodWe analyzed RNA sequences of pretransplant whole blood from subjects followed in three kidney transplant cohorts to identify single nucleotide polymorphisms (SNPs) associated with death censored graft loss (DCGL). We employed a meta-analysis to uncover key transcriptional signatures and pathways associated with the identified SNPs and used single cell RNA to define cellular specificity. We characterized SNP functions using in vitro immunological and survival assays and tested for associations between the identified SNPs and other immune-related diseases using a [~]30,100 subject, electronic health record (EHR)-linked database. ResultsWe uncovered a cluster of four consecutive missense SNPs in the Leukocyte Immunoglobulin-Like Receptor B3 (LILRB3, a negative immune response regulator) gene that strongly associated with DCGL. This LILRB3-4SNPs cluster encodes missense mutations at amino acids 617-618 proximal to a SHP-1/2 phosphatase-binding ITIM motif. LILRB3-4SNPs is specifically enriched within subjects of AA ancestry (8.6% prevalence vs 2.3% in Hispanic and 0.1% in European populations), is not linked to APOL1 G1/G2 alleles, and exhibited a strong association with DCGL. Analysis of PBMC and transplant biopsies from recipients with LILRB3-4SNPs showed evidence of enhanced adaptive immune responsiveness and ferroptosis-associated death in monocytes. Overexpression of the variant allele in THP-1 cells (macrophage line) induced augmented inflammation and ferroptosis, which were attenuated by a ferroptosis inhibitor, verifying a causal link. The LILRB3-4SNPs also associated with multiple systemic and organ-specific immune-related diseases in AAs, consistent with conferring a broadly relevant immune function. Conclusionthe LILRB3-4SNPs represent a functionally important, distinct genetic risk factor for kidney transplant outcome and development/severity of other immune-related diseases in patients of AA ancestry. Pharmacological targeting of ferroptosis should be tested to prevent or treat these disease processes in AA recipients carrying LILRB3-4SNPs.

Variables affecting acquisition and maintenance of operant ethanol self-administration in male and female Long-Evans rats

Wheeler, S. R. — 2024-03-02

AimsThe goal of the present study was to determine the effect of prior experience with ethanol drinking and changes in session duration on the acquisition and maintenance of operant ethanol self-administration. MethodsAdult male and female Long-Evans rats were trained to operantly self-administer ethanol. A subset of male rats underwent intermittent-access two-bottle choice drinking in the home cage prior to operant training. Controls were given access to two bottles of water. Once fully trained in 30-min operant sessions, session duration was reduced to 15 min for all male and female rats. Differences between 30- and 15-min sessions were also assessed in a separate group of male and female rats trained to self-administer sucrose. ResultsNo differences were observed in acquisition rates, the magnitude of responding for ethanol, or total ethanol consumed between male rats allowed to drink ethanol in the home cage and those that remained ethanol naive prior to operant training. A significant decrease in appetitive and consummatory behaviors was observed in males trained to lever press for either ethanol or sucrose. Females exhibited a similar decrease in operant performance for sucrose, but their behavior was largely unchanged in response to changes in session duration when ethanol was the reinforcer. ConclusionsThese data suggest that the use of prior home cage ethanol drinking as an initiation procedure offers little advantage over no initiation procedure at all. Moreover, reducing operant session duration from 30-min to 15-min has the potential to decrease, rather than increase, levels of ethanol intake. Short summaryIntermittent-access two-bottle choice ethanol drinking offers no advantage as an initiation procedure for operant ethanol self-administration over animals that are ethanol-naive prior to training. In addition, shortening the operant session duration does not increase overall intake or promote binge-like patterns of intake for either ethanol or sucrose reinforcer.

Quantitative Superresolution Imaging of F-Actin in the Cell Body and Cytoskeletal Protrusions Using Phalloidin-Based Single-Molecule Labeling and Localization Microscopy

Gunasekara, H. — 2024-03-06

We present single-molecule labeling and localization microscopy (SMLLM) using dye-conjugated phalloidin to achieve enhanced superresolution imaging of filamentous actin (F-actin). We demonstrate that the intrinsic phalloidin dissociation enables SMLLM in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-based SMLLM are better preservation of cellular structures sensitive to mechanical and shear forces during standard sample preparation and more consistent F-actin quantification at the nanoscale. In a proof-of-concept study, we employed SMLLM to super-resolve F-actin structures in U2OS and dendritic cells (DCs) and demonstrate more consistent F-actin quantification in the cell body and structurally delicate cytoskeletal proportions, which we termed membrane fibers, of DCs compared to direct stochastic optical reconstruction microscopy (dSTORM). Using DC2.4 mouse dendritic cells as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane fibers on the culture glass surface after lipopolysaccharide exposure. While our work demonstrates SMLLM for F-actin, the concept opens new possibilities for protein-specific single-molecule labeling and localization in the same step using commercially available reagents.

GABAergic amacrine cells balance biased chromatic information in the mouse retina

Korympidou, M. M. — 2024-03-12

The retina extracts chromatic information present in an animals environment. In the mouse, the feed-forward, excitatory pathway through the retina is dominated by a chromatic gradient, with green and UV signals primarily processed in the dorsal and ventral retina, respectively. However, at the output of the retina, chromatic tuning is more mixed, suggesting that amacrine cells alter spectral tuning. We genetically targeted the population of 40+ GABAergic amacrine cell types and used two-photon calcium imaging to systematically survey chromatic responses in their dendritic processes. We found that amacrine cells show diverse chromatic responses in different spatial regions of their receptive fields and across the dorso-ventral axis of the retina. Compared to their excitatory inputs from bipolar cells, amacrine cells are less chromatically tuned and less likely to be colour-opponent. We identified 25 functional amacrine cell types that, in addition to their chromatic properties, exhibit distinctive achromatic receptive field properties. A combination of pharmacological interventions and a biologically-inspired deep learning model revealed how lateral inhibition and recurrent excitatory inputs shape chromatic properties of amacrine cells. Our data suggest that amacrine cells balance the strongly biased spectral tuning of excitation in the mouse retina and thereby support increased diversity in chromatic information of the retinal output.

Atypical retinal function in a mouse model of Fragile X syndrome

Vlasits, A. L. — 2024-03-17

Altered function of peripheral sensory neurons is an emerging mechanism for symptoms of autism spectrum disorders. Visual sensitivities are common in autism, but whether differences in the retina might underlie these sensitivities is not well-understood. We explored retinal function in the Fmr1 knockout model of Fragile X syndrome, focusing on a specific type of retinal neuron, the "sustained On alpha" retinal ganglion cell. We found that these cells exhibit changes in dendritic structure and dampened responses to light in the Fmr1 knockout. We show that decreased light sensitivity is due to increased inhibitory input and reduced E-I balance. The change in E-I balance supports maintenance of circuit excitability similar to what has been observed in cortex. These results show that loss of Fmr1 in the mouse retina affects sensory function of one retinal neuron type. Our findings suggest that the retina may be relevant for understanding visual function in Fragile X syndrome.

Natural variation in neuraminidase activity influences the evolutionary potential of the seasonal H1N1 lineage hemagglutinin

Liu, T. — 2024-03-18

The antigenic evolution of the influenza A virus hemagglutinin (HA) gene poses a major challenge for the development of vaccines capable of eliciting long-term protection. Prior efforts to understand the mechanisms that govern viral antigenic evolution mainly focus on HA in isolation, ignoring the fact that HA must act in concert with the viral neuraminidase (NA) during replication and spread. Numerous studies have demonstrated that the degree to which the receptor binding avidity of HA and receptor cleaving activity of NA are balanced with each other influences overall viral fitness. We recently showed that changes in NA activity can significantly alter the mutational fitness landscape of HA in the context of a lab-adapted virus strain. Here, we test whether natural variation in relative NA activity can influence the evolutionary potential of HA in the context of the seasonal H1N1 lineage (pdmH1N1) that has circulated in humans since the 2009 pandemic. We observed substantial variation in the relative activities of NA proteins encoded by a panel of H1N1 vaccine strains isolated between 2009 and 2019. We comprehensively assessed the effect of NA background on the HA mutational fitness landscape in the circulating pdmH1N1 lineage using deep mutational scanning and observed pronounced epistasis between NA and residues in or near the receptor binding site of HA. To determine whether NA variation could influence the antigenic evolution of HA, we performed neutralizing antibody selection experiments using a panel of monoclonal antibodies targeting different HA epitopes. We found that the specific antibody escape profiles of HA were highly contingent upon NA background. Overall, our results indicate that natural variation in NA activity plays a significant role in governing the evolutionary potential of HA in the currently circulating pdmH1N1 lineage.

Traumatic injury causes selective degeneration and TDP-43 mislocalization in human iPSC-derived C9orf72-associated ALS/FTD motor neurons

Martin, E. J. — 2024-03-26

A hexanucleotide repeat expansion (HRE) in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, patients with the HRE exhibit a wide disparity in clinical presentation and age of symptom onset suggesting an interplay between genetic background and environmental stressors. Neurotrauma as a result of traumatic brain or spinal cord injury has been shown to increase the risk of ALS/FTD in epidemiological studies. Here, we combine patient-specific induced pluripotent stem cells (iPSCs) with a custom-built device to deliver biofidelic stretch trauma to C9orf72 patient and isogenic control motor neurons (MNs) in vitro. We find that mutant but not control MNs exhibit selective degeneration after a single incident of severe trauma, which can be partially rescued by pretreatment with a C9orf72 antisense oligonucleotide. A single incident of mild trauma does not cause degeneration but leads to cytoplasmic accumulation of TDP-43 in C9orf72 MNs. This mislocalization, which only occurs briefly in isogenic controls, is eventually restored in C9orf72 MNs after 6 days. Lastly, repeated mild trauma ablates the ability of patient MNs to recover. These findings highlight alterations in TDP-43 dynamics in C9orf72 ALS/FTD patient MNs following traumatic injury and demonstrate that neurotrauma compounds neuropathology in C9orf72 ALS/FTD. More broadly, our work establishes an in vitro platform that can be used to interrogate the mechanistic interactions between ALS/FTD and neurotrauma.

Matrimeres are systemic nanoscale mediators of tissue integrity and function

Debnath, K. — 2024-03-29

Tissue barriers must be rapidly restored after injury to promote regeneration. However, the mechanism behind this process is unclear, particularly in cases where the underlying extracellular matrix is still compromised. Here, we report the discovery of matrimeres as constitutive nanoscale mediators of tissue integrity and function. We define matrimeres as non-vesicular nanoparticles secreted by cells, distinguished by a primary composition comprising at least one matrix protein and DNA molecules serving as scaffolds. Mesenchymal stromal cells assemble matrimeres from fibronectin and DNA within acidic intracellular compartments. Drawing inspiration from this biological process, we have achieved the successful reconstitution of matrimeres without cells. This was accomplished by using purified matrix proteins, including fibronectin and vitronectin, and DNA molecules under optimal acidic pH conditions, guided by the heparin-binding domain and phosphate backbone, respectively. Plasma fibronectin matrimeres circulate in the blood at homeostasis but exhibit a 10-fold decrease during systemic inflammatory injury in vivo. Exogenous matrimeres rapidly restore vascular integrity by actively reannealing endothelial cells post-injury and remain persistent in the host tissue matrix. The scalable production of matrimeres holds promise as a biologically inspired platform for regenerative nanomedicine.

Calculating Structure Factors of Protein Solutions by Atomistic Modeling of Protein-Protein Interactions

Qin, S. — 2024-03-28

We present a method, FMAPS(q), for calculating the structure factor, S(q), of a protein solution, by extending our fast Fourier transform-based modeling of atomistic protein-protein interactions (FMAP) approach. The interaction energy consists of steric, nonpolar attractive, and electrostatic terms that are additive among all pairs of atoms between two protein molecules. In the present version, we invoke the free-rotation approximation, such that the structure factor is given by the Fourier transform of the protein center-center distribution function gC(R). At low protein concentrations, gC(R) can be approximated as e-{beta}W(R), where W(R) is the potential of mean force along the center-center distance R. We calculate W(R) using FMAPB2, a member of the FMAP class of methods that is specialized for the second virial coefficient [Qin and Zhou, J Phys Chem B 123 (2019) 8203-8215]. For higher protein concentrations, we obtain S(q) by a modified random-phase approximation, which is a perturbation around the steric-only energy function. Without adjusting any parameters, the calculated structure factors for lysozyme and bovine serum albumin at various ionic strengths, temperatures, and protein concentrations are all in reasonable agreement with those measured by small-angle X-ray or neutron scattering. This initial success motivates further developments, including removing approximations and parameterizing the interaction energy function.

Axonal organelle buildup from loss of AP-4 complex function causes exacerbation of amyloid plaque pathology and gliosis in Alzheimers disease mouse model

Orlowski, A. — 2024-04-01

Lysosomes and related precursor organelles robustly build up in swollen axons that surround amyloid plaques and disrupted axonal lysosome transport has been implicated in worsening Alzheimers pathology. Our prior studies have revealed that loss of Adaptor protein-4 (AP-4) complex function, linked primarily to Spastic Paraplegia (HSP), leads to a similar build of lysosomes in structures we term "AP-4 dystrophies". Surprisingly, these AP-4 dystrophies were also characterized by enrichment of components of APP processing machinery, {beta}-site cleaving enzyme 1 (BACE1) and Presenilin 2. Our studies examining whether the abnormal axonal lysosome build up resulting from AP-4 loss could lead to amyloidogenesis revealed that the loss of AP-4 complex function in an Alzheimers disease model resulted in a strong increase in size and abundance of amyloid plaques in the hippocampus and corpus callosum as well as increased microglial association with the plaques. Interestingly, we found a further increase in enrichment of the secretase, BACE1, in the axonal swellings of the plaques of Alzheimer model mice lacking AP-4 complex compared to those having normal AP-4 complex function, suggestive of increased amyloidogenic processing under this condition. Additionally, the exacerbation of plaque pathology was region-specific as it did not increase in the cortex. The burden of the AP-4 linked axonal dystrophies/AP-4 dystrophies was higher in the corpus callosum and hippocampus compared to the cortex, establishing the critical role of AP-4 -dependent axonal lysosome transport and maturation in regulating amyloidogenic amyloid precursor protein processing. Significance StatementA major pathological feature of Alzheimers disease is the accumulation of axonal lysosomes near sites of amyloid plaques. Lysosome accumulation is thought to contribute to amyloid production. In fact, a genetic perturbation that arrests lysosomes in axons exacerbates amyloid plaque pathology. The mechanisms that control axonal lysosome abundance as well the molecular composition of axonal endolysosomes that produce Abeta, however, are not fully understood. Axonal lysosome build-up is emerging as a common pathology in other neurodegenerative disorders such as Hereditary Spastic Paraplegia (HSP), but its relevance to amyloid production is unknown. We find that a model of HSP caused by loss of AP-4 adaptor complex lead to axonal lysosome buildup that differs in some of its content, but still contributes to amyloidogenesis. This demonstrates that different perturbations leading to changes in heterogeneous pool of axonal lysosomes can converge on a common pathology.

Explorative Discovery of Gene Signatures and Clinotypes in Glioblastoma Cancer Through GeneTerrain Knowledge Map Representation

Saghapour, E. S. — 2024-04-02

This study introduces the GeneTerrain Knowledge Map Representation (GTKM), a novel method for visualizing gene expression data in cancer research. GTKM leverages protein-protein interactions to graphically display differentially expressed genes (DEGs) on a 2-dimensional contour plot, offering a more nuanced understanding of gene interactions and expression patterns compared to traditional heatmap methods. The research demonstrates GTKMs utility through four case studies on glioblastoma (GBM) datasets, focusing on survival analysis, subtype identification, IDH1 mutation analysis, and drug sensitivities of different tumor cell lines. Additionally, a prototype website has been developed to showcase these findings, indicating the methods adaptability for various cancer types. The study reveals that GTKM effectively identifies gene patterns associated with different clinical outcomes in GBM, and its profiles enable the identification of sub-gene signature patterns crucial for predicting survival. The methodology promises significant advancements in precision medicine, providing a powerful tool for understanding complex gene interactions and identifying potential therapeutic targets in cancer treatment.

HOIL1 mediates MDA5 activation through ubiquitination of LGP2

Cheng, D. — 2024-04-03

The RIG-I-like receptors (RLRs), RIG-I and MDA5, are innate sensors of RNA virus infections that are critical for mounting a robust antiviral immune response. We have shown previously that HOIL1, a component of the Linear Ubiquitin Chain Assembly Complex (LUBAC), is essential for interferon (IFN) induction in response to viruses sensed by MDA5, but not for viruses sensed by RIG-I. LUBAC contains two unusual E3 ubiquitin ligases, HOIL1 and HOIP. HOIP generates methionine-1-linked polyubiquitin chains, whereas HOIL1 has recently been shown to conjugate ubiquitin onto serine and threonine residues. Here, we examined the differential requirement for HOIL1 and HOIP E3 ligase activities in RLR-mediated IFN induction. We determined that HOIL1 E3 ligase activity was critical for MDA5-dependent IFN induction, while HOIP E3 ligase activity played only a modest role in promoting IFN induction. HOIL1 E3 ligase promoted MDA5 oligomerization, its translocation to mitochondrial-associated membranes, and the formation of MAVS aggregates. We identified that HOIL1 can interact with and facilitate the ubiquitination of LGP2, a positive regulator of MDA5 oligomerization. In summary, our work identifies LGP2 ubiquitination by HOIL1 in facilitating the activation of MDA5 and the induction of a robust IFN response.

Evidence for intrinsic DNA dynamics and deformability in damage sensing by the Rad4/XPC nucleotide excision repair complex

Baral, S. — 2024-04-03

Altered DNA dynamics at lesion sites are implicated in how DNA repair proteins sense damage within genomic DNA. We examined DNA dynamics related to damage recognition by Rad4 (yeast ortholog of XPC), which recognizes diverse lesions from environmental mutagens and initiates nucleotide excision repair. Using laser temperature-jump (T-jump), we measured the dynamics of DNA containing 3 base-pair mismatches recognized specifically by Rad4 in vitro. The T-jump kinetics traces measured using a cytosine-analog FRET pair, together with rigorous comparison with equilibrium measurements, enabled conformational dynamics to be revealed beyond the T-jump observation window of [~]20 {micro}s - 50 ms. AT-rich nonspecific sites (matched or mismatched) exhibited dynamics primarily within the T-jump window, albeit with some amplitude in "missing" fast (< 20 {micro}s) kinetics. The fast-kinetics amplitudes increased dramatically for specific sites, which were further distinguished by additional missing amplitude in slow (> 50 ms) kinetics at elevated temperatures. We posit that the rapid ({micro}s-ms) fluctuations help stall a diffusing protein at AT-rich/damaged sites and that the >50-ms kinetics reflect a propensity for specific DNA to adopt unwound/bent conformations that may resemble Rad4-bound structures. These studies provide compelling evidence for unusual DNA dynamics and deformability that likely govern how Rad4 senses DNA damage.

Pseudovibriamides from Pseudovibrio marine sponge bacteria promote swarming motility via transcriptional modulation

Dai, Y. — 2024-04-03

Pseudovibrio -Proteobacteria have been repeatedly isolated from marine sponges and proposed to be beneficial to the host. Bacterial motility is known to contribute to host colonization. We have previously identified pseudovibriamides A and B, produced in culture by Pseudovibrio brasiliensis Ab134, and shown that pseudovibriamide A promotes flagellar motility. Pseudovibriamides are encoded in a hybrid nonribosomal peptide synthetase-polyketide synthase gene cluster that also includes several accessory genes. Pseudovibriamide A is a linear heptapeptide and pseudovibriamide B is a nonadepsipeptide derived from pseudovibriamide A. Here we define the borders of the pseudovibriamides gene cluster, assign function to biosynthetic genes using reverse genetics and test the hypothesis that pseudovibriamides impact motility by modulating gene transcription. RNA-seq transcriptomic analyses of strains having different compositions of pseudovibriamides suggested that both pseudovibriamides A and B affect genes potentially involved in motility, and that a compensatory mechanism is at play in mutants that produce only pseudovibriamide A, resulting in comparable swarming motility as the wild type. The data gathered suggest that pseudovibriamides A and B have opposite roles in modulating a subset of genes, with pseudovibriamide B having a primary effect in gene activation, and pseudovibriamide A on inhibition. Finally, we observed many differentially expressed genes (up to 29% of the total gene number) indicating that pseudovibriamides have a global effect on transcription that goes beyond motility. ImportanceMarine sponges are found throughout the oceans from tropical coral reefs to polar sea floors, playing crucial roles in marine ecosystems. Pseudovibrio bacteria have been proposed to contribute to sponge health. We have previously shown that pseudovibriamides produced by Pseudovibrio brasiliensis promote bacterial motility, a behavior that is beneficial to bacterial survival and to host colonization. The gene cluster that encodes pseudovibriamide biosynthesis is found in two thirds of Pseudovibrio genomes. This gene cluster is also present in Pseudomonas bacteria that interact with terrestrial plants and animals. Here we first assign function to pseudovibriamide biosynthetic genes using reverse genetics. We then show that pseudovibriamides play a major role in transcriptional regulation, affecting up to 29% of P. brasiliensis genes, including motility genes. Thus, this work gives insights into pseudovibriamide biosynthesis and provides evidence that they are signaling molecules relevant to bacterial motility and to other yet to be identified phenotypes.

Cyr61 delivery promotes angiogenesis during bone fracture repair

Lang, A. — 2024-04-06

Compromised vascular supply and insufficient neovascularization impede bone repair, increasing risk of non-union. Cyr61, Cysteine-rich angiogenic inducer of 61kD (also known as CCN1), is a matricellular growth factor that is regulated by mechanical cues during fracture repair. Here, we map the distribution of endogenous Cyr61 during bone repair and evaluate the effects of recombinant Cyr61 delivery on vascularized bone regeneration. In vitro, Cyr61 treatment did not alter chondrogenesis or osteogenic gene expression, but significantly enhanced angiogenesis. In a mouse femoral fracture model, Cyr61 delivery did not alter cartilage or bone formation, but accelerated neovascularization during fracture repair. Early initiation of ambulatory mechanical loading disrupted Cyr61-induced neovascularization. Together, these data indicate that Cyr61 delivery can enhance angiogenesis during bone repair, particularly for fractures with stable fixation, and may have therapeutic potential for fractures with limited blood vessel supply.

Harnessing molecular mechanism for precision medicine in dilated cardiomyopathy caused by a mutation in troponin T

Greenberg, L. — 2024-04-09

Familial dilated cardiomyopathy (DCM) is frequently caused by autosomal dominant point mutations in genes involved in diverse cellular processes, including sarcomeric contraction. While patient studies have defined the genetic landscape of DCM, genetics are not currently used in patient care, and patients receive similar treatments regardless of the underlying mutation. It has been suggested that a precision medicine approach based on the molecular mechanism of the underlying mutation could improve outcomes; however, realizing this approach has been challenging due to difficulties linking genotype and phenotype and then leveraging this information to identify therapeutic approaches. Here, we used multiscale experimental and computational approaches to test whether knowledge of molecular mechanism could be harnessed to connect genotype, phenotype, and drug response for a DCM mutation in troponin T, deletion of K210. Previously, we showed that at the molecular scale, the mutation reduces thin filament activation. Here, we used computational modeling of this molecular defect to predict that the mutant will reduce cellular and tissue contractility, and we validated this prediction in human cardiomyocytes and engineered heart tissues. We then used our knowledge of molecular mechanism to computationally model the effects of a small molecule that can activate the thin filament. We demonstrate experimentally that the modeling correctly predicts that the small molecule can partially rescue systolic dysfunction at the expense of diastolic function. Taken together, our results demonstrate how molecular mechanism can be harnessed to connect genotype and phenotype and inspire strategies to optimize mechanism-based therapeutics for DCM. Significance statementDilated cardiomyopathy (DCM), a leading cause of heart failure, is characterized by the inability of the heart to perfuse the body at normal filling pressures. There are multiple causes of DCM, including point mutations in sarcomeric proteins, but most patients receive similar courses of treatment, regardless of the underlying cause of the DCM. Many patients remain unserved by current therapies, and there is a need for new approaches. Here, we use multiscale experimental and computational approaches to demonstrate how knowledge of molecular mechanism can be harnessed to accurately predict the effects of a patient-specific mutation and responses to presumptive therapeutics. Our approach lays the foundation for a precision medicine approach to DCM.

PAMPHLET: A Robust Toolkit for Precise PAM Prediction and Unveiling PAM Consistency in Highly Co-occurrence CRISPR-Cas Systems

Qi, C. — 2024-04-09

The CRISPR-Cas technology has sparked a new technological revolution, significantly enhancing our ability to understand and engineer organisms. The nuclease that underpins this technology is evolving from the "One Cas9 for all" model to a diverse CRISPR toolbox. Identifying PAM sequences is a critical bottleneck in developing novel Cas proteins. Given the limitations of experimental methods, bioinformatics approaches are essential for predicting PAM sequences of Cas proteins in advance. To date, there are only a few PAM sequence prediction programs, and their accuracy is relatively low due to the limited number of spacers in CRISPR-Cas systems. To overcome this challenge, we have developed a pipeline named PAMPHLET, which innovatively utilizes homology searches of Cas proteins to identify additional spacers. PAMPHLET was tested on 20 CRISPR-Cas systems with known PAMs, increasing the number of spacers by up to 18-fold compared to the original datasets and successfully predicting 18 PAM sequences for protospacers. For rigorous and high-quality wet-lab validation of the predictions made by PAMPHLET, we employed the published DocMF platform. This platform leverages next-generation sequencing chips to profile protein-DNA interactions and can simultaneously screen both 5 and 3 PAMs with high throughput. The PAMPHLET predictions showed high consistency with the DocMF results for four novel Cas proteins. We expect that PAMPHLET will overcome the current limitations in PAM sequence prediction, expedite the discovery of PAM sequences, and help to shorten the development cycle for CRISPR tools. Remarkably, PAMPHLET has revealed an intriguing genomic phenomenon: the C2c9 and C2c10 systems, which lack the canonical adaptation module, possess identical PAM sequences to those found in co-occurring type I systems, suggesting potential shared spacer acquisition mechanisms. This finding highlights the complex evolutionary relationships of CRISPR-Cas systems and propels us toward a deeper understanding of their mechanistic diversity and adaptability.

Bridging the Gap: Multi-Omics Profiling of Brain Tissue in Alzheimer's Disease and Older Controls in Multi-Ethnic Populations

Reddy, J. S. — 2024-04-20

INTRODUCTIONMulti-omics studies in Alzheimers disease (AD) revealed many potential disease pathways and therapeutic targets. Despite their promise of precision medicine, these studies lacked African Americans (AA) and Latin Americans (LA), who are disproportionately affected by AD. METHODSTo bridge this gap, Accelerating Medicines Partnership in AD (AMP-AD) expanded brain multi-omics profiling to multi-ethnic donors. RESULTSWe generated multi-omics data and curated and harmonized phenotypic data from AA (n=306), LA (n=326), or AA and LA (n=4) brain donors plus Non-Hispanic White (n=252) and other (n=20) ethnic groups, to establish a foundational dataset enriched for AA and LA participants. This study describes the data available to the research community, including transcriptome from three brain regions, whole genome sequence, and proteome measures. DISCUSSIONInclusion of traditionally underrepresented groups in multi-omics studies is essential to discover the full spectrum of precision medicine targets that will be pertinent to all populations affected with AD.

Geranylgeranylated-SCFFBXO10 Regulates Selective Outer Mitochondrial Membrane Proteostasis and Function

Bhat, S. A. — 2024-04-16

E3-ubiquitin ligases (E3s) are main components of the ubiquitin-proteasome system (UPS), as they determine substrate specificity in response to internal and external cues to regulate protein homeostasis. However, the regulation of membrane protein ubiquitination by E3s within distinct cell membrane compartments or organelles is not well understood. We show that FBXO10, the interchangeable component of the SKP1/CUL1/F-box ubiquitin ligase complex (SCF-E3), undergoes lipid-modification with geranylgeranyl isoprenoid at Cysteine953 (C953), facilitating its dynamic trafficking to the outer mitochondrial membrane (OMM). FBXO10 polypeptide does not contain a canonical mitochondrial targeting sequence (MTS); instead, its geranylgeranylation at C953 and the interaction with two cytosolic factors, PDE6{delta} (a prenyl group-binding protein), and HSP90 (a mitochondrial chaperone) orchestrate specific OMM targeting of prenyl-FBXO10 across diverse membrane compartments. The geranylgeranylation-deficient FBXO10(C953S) mutant redistributes away from the OMM, leading to impaired mitochondrial ATP production, decreased mitochondrial membrane potential, and increased mitochondrial fragmentation. Phosphoglycerate mutase 5 (PGAM5) was identified as a potential substrate of FBXO10 at the OMM using comparative quantitative mass spectrometry analyses of enriched mitochondria (LFQ-MS/MS), leveraging the redistribution of FBXO10(C953S). FBXO10, but not FBXO10(C953S), promoted polyubiquitylation and degradation of PGAM5. Examination of the role of this pathway in a physiological context revealed that the loss of FBXO10 or expression of prenylation-deficient-FBXO10(C953S) inhibited PGAM5 degradation, disrupted mitochondrial homeostasis, and impaired myogenic differentiation of human iPSCs and murine myoblasts. Our studies identify a mechanism for selective E3-ligase mediated regulation of mitochondrial membrane proteostasis and metabolic health, potentially amenable to therapeutic intervention.

In situ cell condensation-based cartilage tissue engineering via immediately implantable high-density stem cell core and rapidly degradable shell microgels

Lee, S. J. — 2024-04-25

Formation of chondromimetic human mesenchymal stem cells (hMSCs) condensations typically required in vitro culture in defined environments. In addition, extended in vitro culture in differentiation media over several weeks is usually necessary prior to implantation, which is costly, time consuming and delays clinical treatment. Here, this study reports on immediately implantable core/shell microgels with a high-density hMSC-laden core and rapidly degradable hydrogel shell. The hMSCs in the core formed cell condensates within 12 hours and the oxidized and methacrylated alginate (OMA) hydrogel shells were completely degraded within 3 days, enabling spontaneous and precipitous fusion of adjacent condensed aggregates. By delivering transforming growth factor-{beta}1 (TGF-{beta}1) within the core, the fused condensates were chondrogenically differentiated and formed cartilage microtissues. Importantly, these hMSC-laden core/shell microgels, fabricated without any in vitro culture, were subcutaneously implanted into mice and shown to form cartilage tissue via cellular condensations in the core after 3 weeks. This innovative approach to form cell condensations in situ without in vitro culture that can fuse together with each other and with host tissue and be matured into new tissue with incorporated bioactive signals, allows for immediate implantation and may be a platform strategy for cartilage regeneration and other tissue engineering applications.

BRWD1 establishes epigenetic states for germinal center initiation, maintenance, and function

Wright, N. E. — 2024-04-28

Germinal center (GC) B cells segregate into three subsets that compartmentalize the antagonistic molecular programs of selection, proliferation, and somatic hypermutation. In bone marrow, the epigenetic reader BRWD1 orchestrates and insulates the sequential stages of cell proliferation and Igk recombination. We hypothesized BRWD1 might play similar insulative roles in the periphery. In Brwd1-/- follicular B cells, GC initiation and class switch recombination following immunization were inhibited. In contrast, in Brwd1-/- GC B cells there was admixing of chromatin accessibility across GC subsets and transcriptional dysregulation including induction of inflammatory pathways. This global molecular GC dysregulation was associated with specific defects in proliferation, affinity maturation, and tolerance. These data suggest that GC subset identity is required for some but not all GC-attributed functions. Furthermore, these data demonstrate a central role for BRWD1 in orchestrating epigenetic transitions at multiple steps along B cell developmental and activation pathways.

GABA and astrocytic cholesterol determine the lipid environment of GABAAR in cultured cortical neurons.

Yuan, Z. — 2024-04-29

The {gamma}-aminobutyric acid (GABA) type A receptor (GABAAR), a GABA activated pentameric chloride channel, mediates fast inhibitory neurotransmission in the brain. The lipid environment is critical for GABAAR function. How lipids regulate the channel in the cell membrane is not fully understood. Here we employed super resolution imaging of lipids to demonstrate that the agonist GABA induces a rapid and reversible membrane translocation of GABAAR to phosphatidylinositol 4,5-bisphosphate (PIP2) clusters in mouse primary cortical neurons. This translocation relies on nanoscopic separation of PIP2 clusters and lipid rafts (cholesterol-dependent ganglioside clusters). In a resting state, the GABAAR associates with lipid rafts and this colocalization is enhanced by uptake of astrocytic secretions. These astrocytic secretions enhance endocytosis and delay desensitization. Our findings suggest intercellular signaling from astrocytes regulates GABAAR location based on lipid uptake in neurons. The findings have implications for treating mood disorders associated with altered neural excitability.

IFI207, a young and fast-evolving protein, controls retroviral replication via the STING pathway

Moran, E. A. — 2024-05-01

Mammalian AIM-2-like receptor (ALR) proteins bind nucleic acids and initiate production of type I interferons or inflammasome assembly, thereby contributing to host innate immunity. In mice, the Alr locus is highly polymorphic at the sequence and copy number level and we show here, is one of the most dynamic regions of the genome. One rapidly evolving gene within this region, Ifi207, was introduced to the Mus genome by gene conversion or an unequal recombination event a few million years ago. Ifi207 has a large, distinctive repeat region that differs in sequence and length among Mus species and even closely related inbred Mus musculus strains. We show that IFI207 controls MLV infection in vivo and that it plays a role in the STING-mediated response to cGAMP, dsDNA, DMXXA and MLV. IFI207 binds to STING and inclusion of its repeat region appears to stabilize STING protein. The Alr locus and Ifi207 provide a clear example of the evolutionary innovation of gene function, possibly as a result of host-pathogen co-evolution. IMPORTANCEThe Red Queen hypothesis predicts that the arms race between pathogens and the host may accelerate evolution of both sides, and therefore cause higher diversity in virulence factors and immune-related proteins, respectively (1). The Alr gene family in mice has undergone rapid evolution in the last few million years and includes the creation of two novel members, MndaL and Ifi207. Ifi207 in particular became highly divergent, with significant genetic changes between highly related inbred mice. IFI207 protein acts in the STING pathway and contributes to anti-retroviral resistance via a novel mechanism. The data show that under the pressure of host-pathogen coevolution in a dynamic locus, gene conversion and recombination between gene family members creates new genes with novel and essential functions that play diverse roles in biological processes.

Reduction of Neuroinflammation and Seizures in a Mouse Model of CLN1 Batten Disease using the Small Molecule Enzyme Mimetic, N-Tert-Butyl Hydroxylamine.

Fyke, Z. — 2024-05-01

Infantile neuronal ceroid lipofuscinosis (CLN1 Batten Disease) is a devastating pediatric lysosomal storage disease caused by pathogenic variants in the CLN1 gene, which encodes the depalmitoylation enzyme, palmitoyl-protein thioesterase 1 (PPT1). CLN1 patients present with visual deterioration, psychomotor dysfunction, and recurrent seizures until neurodegeneration results in death, typically before fifteen years of age. Histopathological features of CLN1 include aggregation of lysosomal autofluorescent storage material (AFSM), as well as profound gliosis. The current management of CLN1 is relegated to palliative care. Here, we examine the therapeutic potential of a small molecule PPT1 mimetic, N-tert-butyl hydroxylamine (NtBuHA), in a Cln1-/- mouse model. Treatment with NtBuHA reduced AFSM accumulation both in vitro and in vivo. Importantly, NtBuHA treatment in Cln1-/- mice reduced neuroinflammation, mitigated epileptic episodes, and normalized motor function. Live cell imaging of Cln1-/- primary cortical neurons treated with NtBuHA partially rescued aberrant synaptic calcium dynamics, suggesting a potential mechanism contributing to the therapeutic effects of NtBuHA in vivo. Taken together, our findings provide supporting evidence for NtBuHA as a potential treatment for CLN1 Batten Disease.

Metabolic immaturity of newborns and breast milk bile acids are the central determinants of heightened neonatal vulnerability to norovirus diarrhea

Peiper, A. M. — 2024-05-01

Noroviruses are the leading global cause of acute gastroenteritis, responsible for 685 million annual cases. While all age groups are susceptible to noroviruses, children are vulnerable to more severe infections than adults, underscored by 200 million pediatric cases and up to 200,000 deaths in children annually. Understanding the basis for the increased vulnerability of young hosts is critical to developing effective treatments. The pathogenic outcome of any enteric virus infection is governed by a complex interplay between the virus, intestinal microbiota, and host immune factors. A central mediator in these complex relationships are host- and microbiota-derived metabolites. Noroviruses bind a specific class of metabolites, bile acids, which are produced by the host and then modified by commensal bacterial enzymes. Paradoxically, bile acids can have both proviral and antiviral roles during norovirus infections. Considering these opposing effects, the microbiota-regulated balance of the bile acid pool may be a key determinant of the pathogenic outcome of a norovirus infection. The bile acid pool in newborns is unique due to immaturity of host metabolic pathways and developing gut microbiota, which could underlie the vulnerability of these hosts to severe norovirus infections. Supporting this concept, we demonstrate herein that microbiota and their bile acid metabolites protect from severe norovirus diarrhea whereas host-derived bile acids promote disease. Remarkably, we also report that maternal bile acid metabolism determines neonatal susceptibility to norovirus diarrhea during breastfeeding by delivering proviral bile acids to the newborn. Finally, directed targeting of maternal and neonatal bile acid metabolism can protect the neonatal host from norovirus disease. Altogether, these data support the conclusion that metabolic immaturity in newborns and ingestion of proviral maternal metabolites in breast milk are the central determinants of heightened neonatal vulnerability to norovirus disease.

Endothelial ERG programs neutrophil transcriptome for sustained anti-inflammatory vascular niche

Vellingiri, V. — 2024-05-05

Neutrophils (PMNs) reside as a marginated pool within the vasculature, ready for deployment during infection. However, how endothelial cells (ECs) control PMN extravasation and activation to strengthen tissue homeostasis remains ill-defined. Here, we found that the vascular ETS-related gene (ERG) is a generalized mechanism regulating PMN activity in preclinical tissue injury models and human patients. We show that ERG loss in ECs rewired PMN-transcriptome, enriched for genes associated with the CXCR2-CXCR4 signaling. Rewired PMNs compromise mice survival after pneumonia and induced lung vascular inflammatory injury following adoptive transfer into naive mice, indicating their longevity and inflammatory activity memory. Mechanistically, EC-ERG restricted PMN extravasation and activation by upregulating the deubiquitinase A20 and downregulating the NF{kappa}B-IL8 cascade. Rescuing A20 in EC-Erg-/- endothelium or suppressing PMN-CXCR2 signaling rescued EC control of PMN activation. Findings deepen our understanding of EC control of PMN-mediated inflammation, offering potential avenues for targeting various inflammatory diseases. HighlightsO_LIERG regulates trans-endothelial neutrophil (PMN) extravasation, retention, and activation C_LIO_LILoss of endothelial (EC) ERG rewires PMN-transcriptome C_LIO_LIAdopted transfer of rewired PMNs causes inflammation in a naive mouse C_LIO_LIERG transcribes A20 and suppresses CXCR2 function to inactivate PMNs C_LI In brief/blurbThe authors investigated how vascular endothelial cells (EC) control polymorphonuclear neutrophil (PMN) extravasation, retention, and activation to strengthen tissue homeostasis. They showed that EC-ERG controls PMN transcriptome into an anti-adhesive and anti-inflammatory lineage by synthesizing A20 and suppressing PMNs-CXCR2 signaling, defining EC-ERG as a target for preventing neutrophilic inflammatory injury.

Rationally Designed Pooled CRISPRi-seq Uncovers a Novel Inhibitor of Bacterial Peptidyl-tRNA Hydrolase

Rahman, A. S. M. Z. — 2024-05-03

Pooled knockdown libraries of essential genes are useful tools for elucidating the mechanisms of action of antibacterial compounds, a pivotal step in antibiotic discovery. However, achieving genomic coverage of antibacterial targets poses a challenge due to the uneven proliferation of knockdown mutants during pooled growth, leading to the unintended loss of important targets. To overcome this issue, we describe the construction of CIMPLE (CRISPRi-mediated pooled library of essential genes), a rationally designed pooled knockdown library built in a model antibiotic-resistant bacteria, Burkholderia cenocepacia. By analyzing growth parameters of clonal knockdown populations of an arrayed CRISPRi library, we predicted strain depletion levels during pooled growth and adjusted mutant relative abundance, approaching genomic coverage of antibacterial targets during antibiotic exposure. We first benchmarked CIMPLE by chemical-genetic profiling of known antibacterials, then applied it to an uncharacterized bacterial growth inhibitor from a new class. CRISPRi-Seq with CIMPLE, followed by biochemical validation, revealed that the novel compound targets the peptidyl-tRNA hydrolase (Pth). Overall, CIMPLE leverages the advantages of arrayed and pooled CRISPRi libraries to uncover unexplored targets for antibiotic action. SummaryBacterial mutant libraries in which antibiotic targets are downregulated are useful tools to functionally characterize novel antimicrobials. These libraries are used for chemical-genetic profiling as target-compound interactions can be inferred by differential fitness of mutants during pooled growth. Mutants that are functionally related to the antimicrobial mode of action are usually depleted from the pool upon exposure to the drug. Although powerful, this method can fail when the unequal proliferation of mutant strains before exposure causes mutants to fall below the detection level in the library pool. To address this issue, we constructed an arrayed essential gene mutant library (EGML) in the antibiotic-resistant bacterium Burkholderia cenocepacia using CRISPR interference (CRISPRi) and analyzed the growth parameters of individual mutant strains. We then modelled depletion levels during pooled growth and used the model to rationally design an optimized CRISPR interference-mediated pooled library of essential genes (CIMPLE). By adjusting the initial inoculum of the knockdown mutants, we achieved coverage of the bacterial essential genome with mutant sensitization. We exposed CIMPLE to a recently discovered antimicrobial of a novel class and discovered it inhibits the peptidyl-tRNA hydrolase, an essential bacterial enzyme. In summary, we demonstrate the utility of CIMPLE and CRISPRi-Seq to uncover the mechanism of action of novel antimicrobial compounds. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/592284v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@b09cf9org.highwire.dtl.DTLVardef@1119fc9org.highwire.dtl.DTLVardef@116b9forg.highwire.dtl.DTLVardef@1b0ce3a_HPS_FORMAT_FIGEXP M_FIG C_FIG

CASTpFold: Computed Atlas of Surface Topography of the universe of protein Folds

Ye, B. — 2024-05-06

Geometric and topological properties of protein structures, including surface pockets, interior cavities, and cross channels, are of fundamental importance for proteins to carry out their functions. Computed Atlas of Surface Topography of proteins (CASTp) is a widely used web server for locating, delineating, and measuring these geometric and topological properties of protein structures. Recent developments in AI-based protein structure prediction such as AlphaFold2 (AF2) have significantly expanded our knowledge on protein structures. Here we present CASTpFold, a continuation of CASTp that provides accurate and comprehensive identifications and quantifications of protein topography. It now provides (i) results on an expanded database of proteins, including the Protein Data Bank (PDB) and non-singleton representative structures of AlphaFold2 structures, covering 183 million AF2 structures; (ii) functional pockets prediction with corresponding Gene Ontology (GO) terms or Enzyme Commission (EC) numbers for AF2-predicted structures; and (iii) pocket similarity search function for surface and protein-protein interface pockets. The CASTpFold web server is freely accessible at https://cfold.bme.uic.edu/castpfold/.

Chronic ethanol exposure produces long-lasting, subregion-specific physiological adaptations in RMTg-projecting mPFC neurons

Przybysz, K. R. — 2024-05-08

Chronic ethanol exposure produces neuroadaptations in the medial prefrontal cortex (mPFC) which facilitate the maladaptive behaviors interfering with recovery from alcohol use disorder. Despite evidence that different cortico-subcortical projections play distinct roles in behavior, few studies have examined the physiological effects of chronic ethanol at the circuit level. The rostromedial tegmental nucleus (RMTg) is a GABAergic midbrain region involved in aversive signaling and is functionally altered by chronic ethanol exposure. Our recent work identified a dense input from the mPFC to the RMTg, yet the effects of chronic ethanol exposure on this circuitry is unknown. In the current study, we examined physiological changes after chronic ethanol exposure in prelimbic (PL) and infralimbic (IL) mPFC neurons projecting to the RMTg. Adult male Long-Evans rats were injected with fluorescent retrobeads into the RMTg and rendered dependent using a 14-day chronic intermittent ethanol (CIE) vapor exposure paradigm. Whole-cell patch-clamp electrophysiological recordings were performed in fluorescently-labeled (RMTg-projecting) and -unlabeled (projection-undefined) layer 5 pyramidal neurons 7-10 days following ethanol exposure. CIE significantly increased intrinsic excitability as well as excitatory and inhibitory synaptic drive in RMTg-projecting IL neurons. In contrast, no lasting changes in excitability were observed in RMTg-projecting PL neurons, although a CIE-induced reduction in excitability was observed in projection-undefined PL neurons. CIE also increased excitatory synaptic drive in RMTg-projecting PL neurons. These data uncover novel subregion- and circuit-specific neuroadaptations in the mPFC following chronic ethanol exposure and reveal that the IL mPFC-RMTg projection is uniquely vulnerable to long-lasting effects of chronic ethanol.

Prefrontal 5α-reductase 2 mediates male-specific acute stress response

Cadeddu, R. — 2024-05-08

A key response to acute stress is the increased brain synthesis of the neurosteroid allopregnanolone (AP). While the rate-limiting step of this reaction is catalyzed by 5-reductase (5R), the role of its two primary isoenzymes, 5R1 and 5R2, in stress reactivity remains unclear. Here, we found that acute stress led to increased levels of 5R2, but not 5R1, in the medial prefrontal cortex (mPFC) of male, but not female, rats. Downregulation of 5R2 in the mPFC significantly reduced stress response in males, and similar sexual dimorphic effects were observed in a novel line of 5R2 knockout rats. Notably, 5R1 regulated baseline AP synthesis, while 5R2 enabled AP production under stress. Acute AP administration restored stress response in 5R2 knockdown rats. Single-nucleus transcriptomics showed that 5R2 enabled stress-induced protein translation in neurons and glia. These results highlight the crucial role of 5R2 in mediating sex-specific differences in acute stress reactivity.

Correlative multiscale 3D imaging of mouse primary and metastatic tumors by sequential light sheet and confocal fluorescence microscopy

Zheng, J. — 2024-05-17

Three-dimensional (3D) optical microscopy, combined with advanced tissue clearing, permits in situ interrogation of the tumor microenvironment (TME) in large volumetric tumors for preclinical cancer research. Light sheet (also known as ultramicroscopy) and confocal fluorescence microscopy are often used to achieve macroscopic and microscopic 3D images of optically cleared tumor tissues, respectively. Although each technique offers distinct fields of view (FOVs) and spatial resolution, the combination of these two optical microscopy techniques to obtain correlative multiscale 3D images from the same tumor tissues has not yet been explored. To establish correlative multiscale 3D optical microscopy, we developed a method for optically marking defined regions of interest (ROIs) within a cleared mouse tumor by employing a UV light-activated visible dye and Z-axis position-selective UV irradiation in a light sheet microscope system. By integrating this method with subsequent tissue processing, including physical ROI marking, reversal of tissue clearing, tissue macrosectioning, and multiplex immunofluorescence, we established a workflow that enables the tracking and 3D imaging of ROIs within tumor tissues through sequential light sheet and confocal fluorescence microscopy. This approach allowed for quantitative 3D spatial analysis of the immune response in the TME of a mouse mammary tumor following cancer immunotherapy at multiple spatial scales. The workflow also facilitated the direct localization of a metastatic lesion within a whole mouse brain. These results demonstrate that our ROI tracking method and its associated workflow offer a novel approach for correlative multiscale 3D optical microscopy, with the potential to provide new insights into tumor heterogeneity, metastasis, and response to therapy at various spatial levels.

"The best home for this paper": A qualitative study of how authors select where to submit manuscripts

Maggio, L. A. — 2024-05-17

IntroductionFor academics selecting a target journal to submit a manuscript is a critical decision with career implications. In medical education, research conducted in 2016 found that authors were influenced by multiple factors such as a journals prestige and its mission. However, since this research was conducted the publishing landscape has shifted to include a broader variety of journals, an increased threat of predatory journals, and new publishing models. This study updates and expands upon how medical education authors decide which journal to submit to with the aim of describing the motivational factors and journal characteristics that guide authors decision making. MethodsThe authors conducted five qualitative focus groups in which twenty-two medical education authors and editors participated. During the focus groups participants were engaged in a discussion about how they select a journal to submit their manuscripts. Audio from all focus groups was transcribed. Transcripts were analyzed using codebook thematic analysis. ResultsParticipants considered multiple factors when selecting a target journal. Factors included a journals impact, the scope of a journal, journal quality, and technical factors (e.g., word limits). Participants also described how social factors influenced their process and that open access plays a role that could both encourage or deter submission. DiscussionThe findings describe the motivational factors and influential signals that guide authors in their journal selection decision making. These findings confirm, extend, and update journal selection factors reported in medical education and other disciplines. Notably, these findings emphasize the role of social factors, relationships and personal experiences, which were absent from previous work. Additionally, we observed increased consideration of OA and a shift away from an emphasis on journal prestige.

Amino Acid-Dependent Material Properties of Tetrapeptide Condensates

Zhang, Y. — 2024-05-15

Condensates formed by intrinsically disordered proteins mediate a myriad of cellular processes and are linked to pathological conditions including neurodegeneration. Rules of how different types of amino acids (e.g., {pi}-{pi} pairs) dictate the physical properties of biomolecular condensates are emerging, but our understanding of the roles of different amino acids is far from complete. Here we studied condensates formed by tetrapeptides of the form XXssXX, where X is an amino acid and ss represents a disulfide bond along the backbone. Eight peptides form four types of condensates at different concentrations and pH values: droplets (X = F, L, M, P, V, A); amorphous dense liquids (X = L, M, P, V, A); amorphous aggregates (X = W), and gels (X = I, V, A). The peptides exhibit enormous differences in phase equilibrium and material properties, including a 368-fold range in the threshold concentration for phase separation and a 3856-fold range in viscosity. All-atom molecular dynamics simulations provide physical explanations of these results. The present work also reveals widespread critical behaviors, including critical slowing down manifested by the formation of amorphous dense liquids and critical scaling obeyed by fusion speed, with broad implications for condensate function.

Paenilamicins from the honey bee pathogen Paenibacillus larvae are context-specific translocation inhibitors of protein synthesis

Koller, T. O. — 2024-05-21

The paenilamicins are a group of hybrid non-ribosomal peptide-polyketide compounds produced by the honey bee pathogen Paenibacillus larvae that display activity against Gram-positive pathogens, such as Staphylococcus aureus. While paenilamicins have been shown to inhibit protein synthesis, their mechanism of action has remained unclear. Here, we have determined structures of the paenilamicin PamB2 stalled ribosomes, revealing a unique binding site on the small 30S subunit located between the A- and P-site tRNAs. In addition to providing a precise description of interactions of PamB2 with the ribosome, the structures also rationalize the resistance mechanisms utilized by P. larvae. We could further demonstrate that PamB2 interferes with the translocation of mRNA and tRNAs through the ribosome during translation elongation, and that this inhibitory activity is influenced by the presence of modifications at position 37 of the A-site tRNA. Collectively, our study defines the paenilamicins as a new class of context-specific translocation inhibitors.

In Vivo Simvastatin and Brain Radiation in a Model of HER2+ Inflammatory Breast Cancer Brain Metastasis

Kumar, S. — 2024-05-30

PurposeInhibiting HMG-CoA reductase with simvastatin prevents breast cancer metastases in preclinical models and radiosensitizes monolayer and stem-like IBC cell lines in vitro. Given the extensive use of simvastatin worldwide and its expected penetration into the brain, we examined whether regulating cholesterol with simvastatin affected IBC3 HER2+ brain metastases. Methods and MaterialsBreast cancer cell lines KPL4 and MDA-IBC3 were examined in vitro for DNA repair after radiation with or without statin treatment. Brain metastasis endpoints were examined in the MDA-IBC3 brain metastasis model after ex vivo exposure to lipoproteins and after tail vein injections with and without whole-brain radiotherapy (WBR) and oral statin exposure. ResultsEx vivo preculture of MDA-IBC3 cells with very low-density lipoprotein (vLDL) enhanced the growth of colonized lesions in the brain in vivo compared with control or high-density lipoprotein (HDL), and concurrent oral simvastatin/ WBR reduced the incidence of micrometastatic lesions evaluated 10 days after WBR. However, statin, with or without WBR, did not reduce the incidence, burden, or number of macrometastatic brain lesions evaluated 5 weeks after WBR. ConclusionsAlthough a role for cholesterol biosynthesis is demonstrated in DNA repair and response to whole brain radiation in this model, durable in vivo efficacy of concurrent whole brain irradiation and oral statin was not demonstrated.

Targeting Unique Ligand Binding Domain Structural Features Downregulates DKK1 in Y537S ESR1 Mutant Breast Cancer Cells

Young, K. S. — 2024-06-02

Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ER) arise after continued therapeutic pressure. Next generation selective estrogen receptor modulators and degraders/downregulators (SERMs and SERDs) show clinical efficacy, but responses are often non-durable. A tyrosine to serine point mutation at position 537 in the ER ligand binding domain (LBD) is among the most common and most pathogenic alteration in this setting. It enables endocrine therapy resistance by superceding intrinsic structural-energetic gatekeepers of ER hormone-dependence, it enhances metastatic burden by enabling neomorphic ER-dependent transcriptional programs, and it resists SERM and SERD inhibiton by reducing their binding affinities and abilities to antagonize transcriptional coregulator binding. However, a subset of SERMs and SERDs can achieve efficacy by adopting poses that force the mutation to engage in a new interaction that favors the therapeutic receptor antagonist conformation. We previously described a chemically unconventional SERM, T6I-29, that demonstrates significant anti-proliferative activities in Y537S ER breast cancer cells. Here, we use a comprehensive suite of structural-biochemical, in vitro, and in vivo approaches to better T6I-29s activities in breast cancer cells harboring Y537S ER. RNA sequencing in cells treated with T6I-29 reveals a neomorphic downregulation of DKK1, a secreted glycoprotein known to play oncogenic roles in other cancers. Importantly, we find that DKK1 is significantly enriched in ER+ breast cancer plasma compared to healthy controls. This study shows how new SERMs and SERDs can identify new therapeutic pathways in endocrine-resistant ER+ breast cancers.

Dietary isoleucine content defines the metabolic and molecular response to a Western diet

Trautman, M. E. — 2024-06-03

The amino acid composition of the diet has recently emerged as a critical regulator of metabolic health. Consumption of the branched-chain amino acid isoleucine is positively correlated with body mass index in humans, and reducing dietary levels of isoleucine rapidly improves the metabolic health of diet-induced obese male C57BL/6J mice. However, it is unknown how sex, strain, and dietary isoleucine intake may interact to impact the response to a Western Diet (WD). Here, we find that although the magnitude of the effect varies by sex and strain, reducing dietary levels of isoleucine protects C57BL/6J and DBA/2J mice of both sexes from the deleterious metabolic effects of a WD, while increasing dietary levels of isoleucine impairs aspects of metabolic health. Despite broadly positive responses across all sexes and strains to reduced isoleucine, the molecular response of each sex and strain is highly distinctive. Using a multi-omics approach, we identify a core sex- and strain-independent molecular response to dietary isoleucine, and identify mega-clusters of differentially expressed hepatic genes, metabolites, and lipids associated with each phenotype. Intriguingly, the metabolic effects of reduced isoleucine in mice are not associated with FGF21 - and we find that in humans plasma FGF21 levels are likewise not associated with dietary levels of isoleucine. Finally, we find that foods contain a range of isoleucine levels, and that consumption of dietary isoleucine is lower in humans with healthy eating habits. Our results demonstrate that the dietary level of isoleucine is critical in the metabolic and molecular response to a WD, and suggest that lowering dietary levels of isoleucine may be an innovative and translatable strategy to protect from the negative metabolic consequences of a WD.

Optimization of systemic AAV9 gene therapy in Niemann-Pick disease type C1 mice

Mylvara, A. V. — 2024-06-08

Niemann-Pick disease, type C1 (NPC1) is a rare, fatal neurodegenerative disorder caused by pathological variants in NPC1, which encodes a lysosomal cholesterol transporter. FDA-approved treatments are limited and do not target the underlying genetic defect. Both systemic and central nervous system (CNS) delivery of AAV9-hNPC1 have shown significant disease amelioration in NPC1 murine models. To assess the impact of dose in null Npc1m1N/m1N mice, we systemically administered three different doses of AAV9-hNPC1 at 4 weeks old. Then, to assess the impact of age, we administered the medium dose before phenotypic onset or at early- or late-stage of disease progression (4, 6 or 8 weeks old, respectively). Higher vector doses and earlier treatment were associated with significantly increased lifespan, slower disease progression, and enhanced CNS transduction. In Npc1I1061T/I1061T mice, a model recapitulating a common human hypomorphic variant, similar benefits ensued. Our findings help define dose ranges, treatment ages, and efficacy in hypomorphic models of NPC1 deficiency and suggest that higher doses of AAV9-hNPC1 in pre-symptomatic disease states are likely to yield better outcomes in NPC1 individuals. Summary BlurbSystemic AAV9-hNPC1 gene therapy in null Npc1m1N mice at higher doses or with earlier administration and treatment of hypomorphic Npc1I1061T mice delays disease progression and increases lifespan.

A New Approach for Discovering Functional Links Connecting Non-Coding Regulatory Variants to Gene Targets

Farooq, H. — 2024-06-14

Genome-wide association studies (GWAS) have linked thousands of genetic variants to various complex traits or diseases. However, most identified variants have weak individual effects, are correlated with nearby polymorphisms due to linkage disequilibrium (LD), and are located in non-coding cis-regulatory elements (CREs). These characteristics complicate the assessment of the direct impact of each variant on tissue specific gene expression and phenotype. To address this challenge, we have developed a novel algorithm that leverages polymer folding and 3D chromatin interactions to prioritize and identify putative causal variants and their target genes. From the millions of eQTL-Gene pairs identified by GTEx in human somatic tissues, we classify only [~]10-20% as putative functional eQTL-Gene pairs supported by phenotypic associations confirmed through CRISPR deletion experiments. Our findings show that unlike most variants, functional eQTL-Gene pairs predominantly reside within the same topologically associating domain (TAD) and have strong associations with cell-type specific cis-regulatory elements (CREs), enriched for binding sites of tissue-specific transcription factors. Unlike most approaches that rely on linear distance or other chromatin features (histone code, accessibility), our algorithm emphasizes the importance of physical interactions and 3D chromatin folding in gene regulation, as the identified eQTL-Gene pairs are all among the small fraction of physical chromatin interactions sufficient for chromatin locus folding. Overall, our algorithm reduces false positive associations between DNA variants and genes identified by eQTL analysis and uncovers novel variant-gene pair associations. These findings suggest a mechanism where a small number of regulatory variants control tissue specific gene expression via their physical association with target genes confined within the same TAD. Our approach provides new insights into the molecular mechanisms driving GWAS phenotypes.

The effects of 17α-estradiol treatment on endocrine system revealed by single-nucleus transcriptomic sequencing of hypothalamus

Li, L. — 2024-06-14

In this study, we investigated 17-estradiols role in lifespan extension from long-term administration. Pooled hypothalami from aged male Norway brown rats treated with 17-estradiol (O.T), aged male controls (O), and young male controls (Y) underwent single-nucleus transcriptomic sequencing (snRNA-seq). To evaluate the effects of 17-estradiol on aging neurons, supervised clustering of neurons based on neuropeptides and their receptors were used to evaluate the responses of each neuron subtype during aging and after 17-estradiol treatment. The elevated cellular metabolism, stress and decreased expression levels of pathways involved in synapse formation in neurons initiated by aging were significantly attenuated by 17-estradiol. Assessment of changes in neuron populations showed that neurons related to food intake, reproduction, blood pressure, stress response, and electrolyte balance were sensitive to 17-estradiol treatment. 17-estradiol treatment not only increased serum Oxytocin (Oxt), but also heightened the activity of hypothalamic-pituitary-gonadal (HPG) axis, as evidenced by significantly elevated levels of plasma Gnrh, total testosterone, and decreased estradiol. Elevated Gnrh1 was confirmed to be one of the causal effects mediating the role of 17-estradiol in energy homeostasis, neural synapse, and stress response. Notably, Crh neurons exhibited prominent stressed phenotype in O.T, distinct to appetite-stimulating neurons Agrp and Ghrl. Therefore, the HPG axis and energy metabolism may be key targets of 17-estradiol in male hypothalamus. Additionally, supervised clustering of neurons was shown to be a useful method for assessing treatment responses and cellular perturbation among different neuron subtypes in the hypothalamus.

Endothelial Drp1 Couples VEGF-induced Redox Signaling with Glycolysis Through Cysteine Oxidation to Drive Angiogenesis

Nagarkoti, S. — 2024-06-16

Angiogenesis plays a vital role for postnatal development and tissue repair following ischemia. Reactive oxygen species (ROS) generated by NADPH oxidases (NOXes) and mitochondria act as signaling molecules that promote angiogenesis in endothelial cells (ECs) which mainly relies on aerobic glycolysis for ATP production. However, the connections linking redox signaling with glycolysis are not well understood. The GTPase Drp1 is a member of the dynamin superfamily that moves from cytosol to mitochondria through posttranslational modifications to induce mitochondrial fission. The role of Drp1 in ROS-dependent VEGF signaling and angiogenesis in ECs has not been previously described. Here, we identify an unexpected function of endothelial Drp1 as a redox sensor, transmitting VEGF-induced H2O2 signals to enhance glycolysis and angiogenesis. Loss of Drp1 expression in ECs inhibited VEGF-induced angiogenic responses. Mechanistically, VEGF rapidly induced the NOX4-dependent sulfenylation (CysOH) of Drp1 on Cys644, promoting disulfide bond formation with the metabolic kinase AMPK and subsequent sulfenylation of AMPK at Cys299/304 via the mitochondrial fission-mitoROS axis. This cysteine oxidation of AMPK, in turn, enhanced glycolysis and angiogenesis. In vivo, mice with EC-specific Drp1 deficiency or CRISPR/Cas9-engineered "redox-dead" (Cys to Ala) Drp1 knock-in mutations exhibited impaired retinal angiogenesis and post-ischemic neovascularization. Our findings uncover a novel role for endothelial Drp1 in linking VEGF-induced mitochondrial redox signaling to glycolysis through a cysteine oxidation-mediated Drp1-AMPK redox relay, driving both developmental and reparative angiogenesis.

AP2/ERF transcription factors enriched in the drought-response transcriptome of the Thar desert tree Prosopis cineraria show higher copy number and greater DNA-binding affinity than orthologs in drought-sensitive species

Dhiman, V. — 2024-06-19

We sequenced the drought-response transcriptome of the keystone tree species Prosopis cineraria from the Indian Thar desert to understand the key factors in its drought tolerance mechanism. We identified a network of genes activated in P. cineraria involved in osmotic stress response, phytohormone, calcium, and phosphorelay signal transduction. Of these, up-regulation of 54 APETALA2/Ethylene-Responsive Factor (AP2/ERF) transcription factor genes, validated by real-time PCR, suggests their key role in the drought tolerance of P. cineraria. We conducted a genome-wide study of the AP2/ERF superfamily in P. cineraria, classifying its 232 proteins into 15 clades and analyzing their protein structures, gene structure, and promoter organization. The P. cineraria genome contains more copies of AP2/ERF genes than drought-sensitive plants. Further, we identified sequence polymorphisms in AP2/ERF genes between Arabian and Indian cultivars of P. cineraria. We modeled the DNA-protein complex structures of AP2/ERFs from drought-tolerant and sensitive species using AlphaFold to compare their DNA binding ability. Though the DNA binding domain (DBD) is relatively conserved across species, the unstructured region of these proteins possesses different charge distributions, which might contribute differently to their DNA search and binding. Using all-atom molecular dynamics simulations, we teased out a higher number of specific DBD-DNA hydrogen bonds in P. cineraria, leading to a stronger DNA-binding affinity compared to drought-sensitive Arabidopsis thaliana. These results directly support copy number expansion of AP2/ERF transcription factors and the evolution of their structures for more efficient DNA search and binding as drought adaptation mechanisms in P. cineraria.

Top-Down Proteomics Identifies Plasma Proteoform Signatures of Liver Cirrhosis Progression

Forte, E. — 2024-06-19

Cirrhosis, advanced liver disease, affects 2-5 million Americans. While most patients have compensated cirrhosis and may be fairly asymptomatic, many decompensate and experience life-threatening complications such as gastrointestinal bleeding, confusion (hepatic encephalopathy), and ascites, reducing life expectancy from 12 to less than 2 years. Among patients with compensated cirrhosis, identifying patients at high risk of decompensation is critical to optimize care and reduce morbidity and mortality. Therefore, it is important to preferentially direct them towards specialty care which cannot be provided to all patients with cirrhosis. We used discovery Top-down Proteomics (TDP) to identify differentially expressed proteoforms (DEPs) in the plasma of patients with progressive stages of liver cirrhosis with the ultimate goal to identify candidate biomarkers of disease progression. In this pilot study, we identified 209 DEPs across three stages of cirrhosis (compensated, compensated with portal hypertension, and decompensated), of which 115 derived from proteins enriched in the liver at a transcriptional level and discriminated the three stages of cirrhosis. Enrichment analyses demonstrated DEPs are involved in several metabolic and immunological processes known to be impacted by cirrhosis progression. We have preliminarily defined the plasma proteoform signatures of cirrhosis patients, setting the stage for ongoing discovery and validation of biomarkers for early diagnosis, risk stratification, and disease monitoring. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/599662v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@797e27org.highwire.dtl.DTLVardef@495bb7org.highwire.dtl.DTLVardef@2478a9org.highwire.dtl.DTLVardef@6dbc0a_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPerformed a pilot top-down LC-MS/MS analysis to identify proteoforms (PFRs) in the plasma of patients with 3 progressive stages of liver cirrhosis. C_LIO_LIIdentified 2867 proteoforms (PFRs) and 209 differentially regulated proteoforms (DRPs) in the different stages of the disease. C_LIO_LIIdentified DRP profiles able to potentially distinguish early from late stages of the disease, including 115 liver-derived DRPs. C_LIO_LIFibrinogen alpha chain, haptoglobin, and Apo A-I are the proteins with the highest number of DRPs and represent potential candidate biomarkers of liver cirrhosis progression. C_LI

Epigenetic changes regulating the epithelial-mesenchymal transition in human trophoblast differentiation

Ackerman, W. E. — 2024-07-04

The phenotype of human placental extravillous trophoblast (EVT) at the end of pregnancy reflects both first trimester differentiation from villous cytotrophoblast (CTB) and later gestational changes, including loss of proliferative and invasive capacity. Invasion abnormalities are central to two major placental pathologies, preeclampsia and placenta accreta spectrum, so characterization of the corresponding normal processes is crucial. In this report, our gene expression analysis, using purified human CTB and EVT cells, highlights an epithelial- mesenchymal transition (EMT) mechanism underlying CTB-EVT differentiation and provides a trophoblast-specific EMT signature. In parallel, DNA methylation profiling shows that CTB cells, already hypomethylated relative to non-trophoblast cell lineages, show further genome- wide hypomethylation in the transition to EVT. However, a small subgroup of genes undergoes gains of methylation (GOM) in their regulatory regions or gene bodies, associated with differential mRNA expression (DE). Prominent in this GOM-DE group are genes involved in the EMT, including multiple canonical EMT markers and the EMT-linked transcription factor RUNX1, for which we demonstrate a functional role in modulating the migratory and invasive capacities of JEG3 trophoblast cells. This analysis of DE associated with locus-specific GOM, together with functional studies of an important GOM-DE gene, highlights epigenetically regulated genes and pathways acting in human EVT differentiation and invasion, with implications for obstetric disorders in which these processes are dysregulated.

Chemogenetic control of GABAergic activity within the interpeduncular nucleus reveals dissociable behavioral components of the nicotine withdrawal phenotype

Miguelez Fernandez, A. M. M. — 2024-07-10

Chronic exposure to nicotine results in the development of a dependent state such that a withdrawal syndrome is elicited upon cessation of nicotine. The habenulo-interpeduncular (Hb-IPN) circuit contains a high concentration of nAChRs and has been identified as a main circuit involved in nicotine withdrawal. Here we investigated the contribution of two distinct subpopulations of IPN GABAergic neurons to nicotine withdrawal behaviors. Using a chemogenetic approach to specifically target Amigo1-expressing or Epyc-expressing neurons within the IPN, we found that activity of the Amigo1 and not the Epyc subpopulation of GABAergic neurons is critical for anxiety-like behaviors both in naive mice and in those undergoing nicotine withdrawal. Moreover, data revealed that stimulation of Amigo1 neurons in nicotine-naive mice elicits opposite effects on affective and somatic signs of withdrawal. Taken together, these results suggest that somatic and affective behaviors constitute dissociable components of the nicotine withdrawal phenotype and are likely supported by distinct subpopulations of neurons within the IPN.

Star-polymers as potent broad-spectrum extracellular virucidal antivirals

Super, E. H. — 2024-07-10

Viruses pose a significant threat to both global health and the global economy. It is clear that novel antiviral strategies are urgently needed, with a broad-spectrum approach being most desired. We have discovered a broad-spectrum, non-toxic polymer virucide that can tackle the viral threat. This polymeric virucide is effective at nanomolar concentrations, against a broad-spectrum of viruses and, demonstrated using an intranasal respiratory syncytial virus (RSV) murine model, has excellent efficacy, low anti-coagulant properties and low toxicity in vivo. Molecular dynamic simulations show that this polymer achieves its virucidal antiviral effect via self-assembly of viral-receptors leading to increased envelope forces and viral disassembly. The discovery of this cheap and readily produced polymer marks the start of a new type of receptor-crosslinking broad-spectrum antiviral that has significant potential to combat the global threat posed by viruses.

Context-dependent roles for autophagy in myeloid cells in tumor progression

Choi, J. — 2024-07-16

Autophagy is known to suppress tumor initiation by removing genotoxic stresses in normal cells. Conversely, autophagy is also known to support tumor progression by alleviating metabolic stresses in neoplastic cells. Centered on this pro-tumor role of autophagy, there have been many clinical trials to treat cancers through systemic blocking of autophagy. Such systemic inhibition affects both tumor cells and non-tumor cells, and the consequence of blocked autophagy in non-tumor cells in the context of tumor microenvironment is relatively understudied. Here, we examined the effect of autophagy-deficient myeloid cells on the progression of autophagy-competent tumors. We found that blocking autophagy only in myeloid cells modulated tumor progression markedly but such effects were context dependent. In a tumor implantation model, the growth of implanted tumor cells was substantially reduced in mice with autophagy-deficient myeloid cells; T cells infiltrated deeper into the tumors and were responsible for the reduced growth of the implanted tumor cells. In an oncogene-driven tumor induction model, however, tumors grew faster and metastasized more in mice with autophagy- deficient myeloid cells. These data demonstrate that the autophagy status of myeloid cells plays a critical role in tumor progression, promoting or suppressing tumor growth depending on the context of tumor-myeloid cell interactions. This study indicates that systemic use of autophagy inhibitors in cancer therapy may have differential effects on rates of tumor progression in patients due to effects on myeloid cells and that this warrants more targeted use of selective autophagy inhibitors in a cancer therapy in a clinical setting.

Modelling alcohol consumption in rodents using two-bottle choice home cage drinking and optional lickometry-based microstructural analysis

Hou, S. — 2024-07-23

Two-bottle choice home cage drinking is one of the most widely used paradigms to study ethanol consumption in rodents. In its simplest form, animals are provided with access to two drinking bottles, one of which contains regular tap water and the other ethanol, for 24 hr/day with daily intake measured via change in bottle weight over the 24 hr period. Consequently, this approach requires no specialized laboratory equipment. While such ease of implementation is likely the greatest contributor to its widespread adoption by preclinical alcohol researchers, the resolution of drinking data acquired using this approach is limited by the number of times the researcher measures bottle weight (e.g., once daily). However, the desire to examine drinking patterns in the context of overall intake, pharmacological interventions, and neuronal manipulations has prompted the development of home cage lickometer systems that can acquire data at the level of individual licks. Although a number of these systems have been developed recently, the open-source system, LIQ HD, has garnered significant attention in the field for its affordability and user friendliness. Although exciting, this system was designed for use in mice. Here, we review appropriate procedures for standard and lickometer-equipped two-bottle choice home cage drinking. We also introduce methods for adapting the LIQ HD system to rats including hardware modifications to accommodate larger cage size and a redesigned 3D printed bottle holder compatible with standard off-the-shelf drinking bottles. Using this approach, researchers can examine daily drinking patterns in addition to levels of intake in many rats in parallel thereby increasing the resolution of acquired data with minimal investment in additional resources. These methods provide researchers with the flexibility to use either standard bottles or a lickometer-equipped apparatus to interrogate the neurobiological mechanisms underlying alcohol drinking depending on their precise experimental needs. SUMMARYThis protocol describes a standard intermittent-access two-bottle choice home cage drinking paradigm to model alcohol consumption in rats. In addition, it provides step-by-step instructions to augment the standard protocol with a DIY lickometer system that enables microstructural analysis of drinking behavior.

Evaluation of Methodologies in Anti-nephrin Autoantibody Detection

Liu, P. — 2024-07-25

Recent studies discovered the prominent presence of anti-nephrin autoantibodies in minimal change disease, steroid-sensitive nephrotic syndrome and/or post-transplant recurrent focal segmental glomerulosclerosis (FSGS). However, widely different, and often unconventional autoantibody detection methods were used among these studies, making it challenging to assess the pathogenic role for the antibodies. Here we examined methods of conventional ELISA, magnetic on-beads ELISA, immunoprecipitation-immunoblotting (IP-IB), and cell- and tissue-based antibody assays with 127 plasma samples of kidney and non-kidney diseases. On the antigen side, we compared commercially available recombinant human nephrin extracelluar domain (ECD) produced from human or mouse cell lines, as well as lab-made full length, ECD, and series of ECD truncates for measuring autoantibody reactivity and specificity. Surprisingly, different assay methods and different antigen preparations led to observation of assay-specific false-positive and false-negative results. In general, a set of tests that combines magnetic beads-enhanced ELISA, followed by IP-IB, and epitope mapping showed the most robust results for anti-nephrin autoantibodies, detected in two primary FSGS patients among all cases tested. It is interesting to note that cell/tissue-based results, also supported by antigen truncation studies, clearly suggest steric hindrance of reactive epitopes, as in full length nephrin that forms compact self-associated complexes. In conclusion, anti-nephrin positivity is rare among the tested patients (2/127), including those with FSGS (2/42), and autoantibody results can be affected by the choice of detection methods.

CD11b suppresses TLR7-driven inflammatory signaling to protect against lupus nephritis

Li, X. — 2024-07-26

Lupus Nephritis (LN) is a severe complication of systemic lupus erythematosus (SLE) that affects kidney function. Here, we investigated the role of CD11b, a protein encoded by the ITGAM gene, in the development of LN and its functional activation as a therapeutic strategy. Genetic coding variants of ITGAM significantly increase the risk for SLE and LN by producing a less active CD11b and leading to elevated levels of type I interferon (IFN I). However, a molecular mechanism for how these variants increase LN risk has been unclear. Here, we determined that these variants also significantly associate with elevations in soluble urokinase plasminogen activator receptor (suPAR), a known biomarker linked to kidney disease, suggesting a novel molecular connection. Pharmacologic activation of CD11b with a novel, clinical-stage agonist ONT01 significantly suppressed suPAR production in myeloid cells and reduced systemic inflammation and kidney damage in multiple experimental models of LN. Importantly, delaying treatment with ONT01 until after disease onset also significantly reduced serum suPAR and inflammatory cytokines, and decreased immune complex deposition in the glomerulus, glomerulonephritis and albuminuria, suggesting that CD11b activation is therapeutic for LN. Genetic activation of CD11b via a gain-of-function CD11b mutation also showed complete protection from LN, whereas genetic deletion of CD11b worsened the disease in mice, providing further evidence of the role of CD11b activation in regulating LN. Finally, transfer of human LN PBMCs generated human LN like disease in mice that was significantly reduced by ONT01. Together, these data provide strong evidence that ONT01 mediated CD11b activation can therapeutically modulate TLR7-driven inflammation and protect against LN. These findings support clinical development of CD11b agonists as novel therapeutics for treating lupus nephritis in human patients.

ARPE-19 Epithelial Cells Fail To Initiate Type-I Interferon Signaling in Response to Human Cytomegalovirus Infection.

Andrade-Medina, M. — 2024-08-01

ARPE-19 cells are a commonly used epithelial model for studying human cytomegalovirus (HCMV) infection. We recently found that ARPE-19 cells assume a mesenchymal phenotype when maintained at low confluency and that ARPE-19 cells resemble mesenchymal fibroblasts rather than epithelial cells in HCMV infection assays. Here, using comparative proteomics analysis, we find that subconfluent ARPE-19 cells are also deficient in their ability to initiate canonical type-I interferon signaling. Comparative proteomic analysis between subconfluent ARPE-19 and MRC-5 cells revealed a lack of canonical type-I interferon response in ARPE-19 cells upon HCMV infection, evidenced by the absence of interferon stimulated gene (ISG) induction. qRT-PCR and RNA-sequencing analysis revealed that ARPE-19 cells fail to initiate interferon-beta transcription in response to HCMV infection, yet they are competent to respond to exogenously interferon-b, indicating a failure in early pathogen detection. ARPE-19 cells showed low baseline levels of key intracellular pattern recognition receptors (PRRs) such as CGAS and IFI16, as well as the signaling molecule STING. This deficiency was associated with a failure to activate IRF3 phosphorylation, a crucial step in interferon signaling. These findings suggest an upstream defect in the early detection of viral components, likely due to reduced expression of critical PRRs. ARPE-19 cells may be inherently deficient in initiating interferon responses due to their derivation or possibly due to their origin from an immune-privileged tissue. Our results continue to highlight important phenotypic characteristics of the ARPE-19 cell line; important considerations for those using ARPE-19 cells as an experimental infection model for studying HCMV or other human viruses.

Evaluation of vectors for gene expression in Pseudovibrio bacteria and their application in Aplysina marine sponge studies

Dai, Y. — 2024-08-02

-Proteobacteria belonging to the Pseudovibrio genus have been isolated from different marine organisms including marine sponges, corals and algae. This genus was first described in 2004 and has since garnered attention due to the potential ecological relevance and biotechnological application of its metabolites. For instance, we recently reported specialized metabolites we named pseudovibriamides from Pseudovibrio brasiliensis Ab134. The pseudovibriamide encoding ppp gene cluster is found in two thirds of Pseudovibrio genomes. Pseudovibriamides coordinate motility and biofilm formation, behaviors that are known to be important for host colonization. Although we previously established reverse genetics methods to delete genes via homologous recombination, no self-replicative vectors have been reported for Pseudovibrio. We show that plasmid vectors containing two different broad-host-range replicons, RSF1010 and pBBR1, can be used in P. brasiliensis. The efficiency of vector transfer by electroporation averaged [~] 3 x 103 CFU/{micro}g plasmid DNA whereas the conjugation frequency from E. coli ranged from 10-3 to 10-6. We then tested the vectors for fluorescent protein expression and consequent labeling, which allowed us to observe their effects on swarming motility and to compare plasmid stability. This study expands the genetic toolbox available for Pseudovibrio which is expected to enable future ecological and biotechnological studies. ImportanceThe genus Pseudovibrio of -Proteobacteria has consistently been isolated from marine sponges and other marine organisms such as corals and algae. Pseudovibrio bacteria are a source of antibiotics and other secondary metabolites with the potential to be developed into pharmaceuticals. Moreover, the secondary metabolites they produce are important for their physiology and for interactions with other organisms. Here we expand the genetic tool box available for Pseudovibrio bacteria by establishing self-replicative vectors that can be used for the expression of e.g., fluorescent proteins. The availability of genetic tools is important to enable us to explore the emerging ecological and biotechnological potential of Pseudovibrio bacteria.

Sulfoglycodendron Antivirals with Scalable Architectures and Activities

KRAL, P. — 2024-08-02

Many viruses initiate their cell-entry by binding their multi-protein receptors to human heparan sulfate proteoglycans (HSPG) and other molecular components present on cellular membranes. These viral interactions could be blocked and the whole viruses could be eliminated by suitable HSPG-mimetics providing multivalent binding to viral protein receptors. Here, large sulfoglycodendron HSPG-mimetics of different topologies, structures, and sizes were designed to this purpose. Atomistic molecular dynamics simulations were used to examine the ability of these broad-spectrum antivirals to block multi-protein HSPG-receptors in HIV, SARS-CoV-2, HPV, and dengue viruses. To characterize the inhibitory potential of these mimetics, their binding to individual and multiple protein receptors was examined. In particular, vectorial distributions of binding energies between the mimetics and viral protein receptors were introduced and calculated along the simulated trajectories. Space-dependent residual analysis of the mimetic-receptor binding was also performed. This analysis revealed detail nature of binding between these antivirals and viral protein receptors, and provided evidence that large inhibitors with multivalent binding might act like a molecular glue initiating the self-assembly of protein receptors in enveloped viruses. TOC FIGURE O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/606251v2_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@55801borg.highwire.dtl.DTLVardef@14dac62org.highwire.dtl.DTLVardef@707fa1org.highwire.dtl.DTLVardef@1cb451a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Along- and cross-muscle fiber shear moduli in skeletal muscle

Sahani, R. — 2024-08-03

The material properties of muscle play a central role in how muscle resists joint motion, transmits forces internally, and repairs itself. While many studies have evaluated muscles tensile material properties, few have investigated muscles shear properties. The objective of this study was to quantify the shear moduli of skeletal muscle both along (along-muscle fiber) and perpendicular (cross-muscle fiber) to the direction of muscle fibers. We collected data from the extensor digitorum longus, tibialis anterior, and soleus muscles harvested from both hindlimbs of 12 rats. These muscles were chosen to further evaluate the consistency of shear moduli across muscles with different architectures. We applied strains and measured stress in three configurations: parallel, perpendicular, and across the muscle fibers to characterize the along- and cross-muscle fiber tensile and shear material parameters. We found no significant difference between the shear modulus measured parallel to the fibers (along-muscle fiber) and the shear modulus in the plane perpendicular to the fibers (cross-muscle fiber). Although the shear moduli were not significantly different, there was a greater difference with increasing strain, suggesting that there is greater anisotropy at larger strains. We also found no significant difference in moduli between the muscles with differing muscle architecture. These results characterize the shear behavior of skeletal muscle and are relevant to understanding the role of shear in force transmission and injury.

The Epithelial-to-Mesenchymal Transition Supports HCMV Infection and Biosynthesis in Mammary Epithelial Cells Through Two Distinct Mechanisms.

Golconda, P. — 2024-08-12

Human cytomegalovirus (HCMV) infects a wide range of cell types in the body, including a variety of epithelial cell types. Despite the significance of epithelial cells during infection, HCMV has been difficult to study in epithelial cells. In this study, we examined HCMV infection in mammary and prostate epithelial cell lines, finding that the virus establishes a semi-permissive, biosynthetically abortive state. Building on previous work, we hypothesized that shifting epithelial cells to a mesenchymal cell state would restore HCMV biosynthesis and progeny production. To test this hypothesis, we induced epithelial-to-mesenchymal transition (EMT) using TGF-{beta} and the EMT-transcription factor (EMT-TF) SNAIL. We found that shifting strongly epithelial cell lines to a mesenchymal cell state shifted HCMV infection from a semi-permissive to fully permissive state. This effect appeared to involve two distinct mechanisms: EMT-sensitive enhancement of viral entry and EMT-sensitive enhancement of viral mRNA translation. Although the precise mechanisms remain elusive, our findings identify the epithelial-mesenchymal cell state axis as an important regulator of HCMV infection and provide new insights into how cellular differentiation states influence viral replication. They also raise the possibility that the EMT pathway, a fundamental pathway involved in development and cancer metastasis, could regulate HCMV infection in-vivo, potentially contributing to viral persistence or pathogenesis in epithelial tissues.

DHX15 inhibits mouse APOBEC3 deamination

Zhao, W. — 2024-08-19

APOBEC3 family proteins are critical host factors that counteract and prevent the replication of retroviruses and other viruses through cytidine deamination. Human APOBEC3 proteins inactivate HIV-1 through the introduction of lethal mutations to viral genomes. In contrast, mouse APOBEC3 does not induce DNA hypermutation of murine retroviruses, although it retains functional cytidine deaminase activity. Why mouse APOBEC3 does not effectively deaminate murine retroviruses is still unknown. In this study, we found that the dead box helicase DHX15 interacts with mouse APOBEC3 and inhibits its deamination activity. DHX15 was packaged into murine leukemia virus (MLV) virions independent of its binding with APOBEC3. Moreover, DHX15 knockdown inhibited MLV replication and resulted in more G-to-A mutations in proviral DNA. Finally, DHX15 knockdown induced DNA damage in murine cells, suggesting that it plays a role in preserving genome integrity in cells expressing mouse APOBEC3 protein.

Altered Copper Transport in Oxidative Stress-Dependent Brain Endothelial Barrier Dysfunction Associated with Alzheimer's Disease

Hossain, M. S. — 2024-08-29

Oxidative stress and blood-brain barrier (BBB) disruption due to brain endothelial barrier dysfunction contribute to Alzheimers Disease (AD), which is characterized by beta-amyloid (A{beta}) accumulation in senile plaques. Copper (Cu) is implicated in AD pathology and its levels are tightly controlled by several Cu transport proteins. However, their expression and role in AD, particularly in relation to brain endothelial barrier function remains unclear. In this study, we examined the expression of Cu transport proteins in the brains of AD mouse models as well as their involvement in A{beta}42-induced brain endothelial barrier dysfunction. We found that the Cu uptake transporter CTR1 was upregulated, while the Cu exporter ATP7A and/or ATP7B were downregulated in the hippocampus of AD mouse models, and in A{beta}42-treated human brain microvascular endothelial cells (hBMECs). In the 5xFAD AD mouse model, Cu levels (assessed by ICP-MS) were elevated in the hippocampus. Moreover, A{beta}42-induced reactive oxygen species (ROS) production, ROS-dependent loss in barrier function in hBMEC (measured by transendothelial electrical resistance), and tyrosine phosphorylation of VE-cadherin were all inhibited by either a membrane permeable Cu chelator or by knocking down CTR1 expression. These findings suggest that dysregulated expression of Cu transport proteins may lead to intracellular Cu accumulation in the AD brain, and that A{beta}42 promotes ROS-dependent brain endothelial barrier dysfunction and VE-Cadherin phosphorylation in a CTR1-Cu-dependent manner. Our study uncovers the critical role of Cu transport proteins in oxidative stress-related loss of BBB integrity in AD. HighlightsO_LIUpregulation of the Cu importer CTR1 and downregulation of the Cu exporter ATP7A in the hippocampus of AD mouse models C_LIO_LIA{beta}42 increases CTR1 expression while reduces ATP7A and ATP7B levels in human brain microvascular ECs. C_LIO_LIA{beta}42 triggers increased reactive oxygen species (ROS) production in human brain microvascular ECs through a CTR1- and Cu-dependent manner. C_LIO_LIA{beta}42 induces endothelial barrier dysfunction in human brain microvascular ECs through a CTR1-Cu-ROS-pendent manner. C_LI

From impact metrics and open science to communicating research: Journalists' awareness of academic controversies

Fleerackers, A. — 2024-09-04

This study sheds light on how journalists respond to evolving debates within academia around topics including research integrity, improper use of metrics to measure research quality and impact, and the risks and benefits of the open science movement. Drawing on semi-structured interviews with 19 health and science journalists, we describe journalists awareness of these controversies and the ways in which that awareness, in turn, shapes the practices they use to select, verify, and communicate research. Our findings suggest that journalists perceptions of debates in scholarly communication vary widely, with some displaying a highly critical and nuanced understanding and others presenting a more limited awareness. Those with a more in-depth understanding report closely scrutinizing the research they report, carefully vetting the study design, methodology, and analyses. Those with a more limited awareness are more trusting of the peer review system as a quality control system and more willing to rely on researchers when determining what research to report on and how to vet and frame it. We discuss the benefits and risks of these varied perceptions and practices, highlighting the implications for the nature of the research media coverage that reaches the public.

Membrane-assisted Aβ40 aggregation pathways

Muhammedkutty, F. N. K. — 2024-09-08

Alzheimers disease (AD) is caused by the assembly of amyloid-beta (A{beta}) peptides into oligomers and fibrils. Endogenous A{beta} aggregation may be assisted by cell membranes, which can accelerate the nucleation step enormously, but knowledge of membrane-assisted aggregation is still very limited. Here we used extensive MD simulations to structurally and energetically characterize key intermediates along the membrane-assisted aggregation pathways of A{beta}40. Reinforcing experimental observations, the simulations reveal unique roles of GM1 ganglioside and cholesterol in stabilizing membrane-embedded {beta}-sheets and of Y10 and K28 in the ordered release of a small oligomeric seed into solution. The same seed leads to either an open-shaped or R-shaped fibril, with significant stabilization provided by inter- or intra-subunit interfaces between a straight {beta}- sheet (residues Q15-D23) and a bent {beta}-sheet (residues A30-V36). This work presents the first comprehensive picture of membrane-assisted aggregation of A{beta}40, with broad implications for developing AD therapies and rationalizing disease-specific polymorphisms of amyloidogenic proteins.

Presence of distinct operant phenotypes and transient withdrawal-induced escalation of operant ethanol intake in female rats

Pitock, J. R. — 2024-09-10

Operant self-administration is frequently used to investigate the neurobiological mechanisms underlying alcohol seeking and drinking and to test the efficacy of drugs under development for the treatment of alcohol use disorder (AUD). Although widely used by the research community, there is a paucity of operant ethanol self-administration studies that include female subjects. The current study characterizes home cage drinking and operant ethanol self-administration in female Sprague Dawley, Long Evans, and Wistar rats. Rats underwent three weeks of intermittent-access two-bottle choice home cage drinking before being trained to lever press for ethanol in standard operant chambers equipped with contact lickometers. After capturing baseline operant performance, rats were chronically exposed to control or ethanol liquid diet using the Lieber-DeCarli method. Operant ethanol self-administration was re-evaluated after chronic liquid diet exposure to determine whether female rats exhibit similar withdrawal-induced escalation of ethanol intake as is regularly observed in male rats. Our findings reveal the presence of three distinct operant phenotypes (Drinker, Responder, Nonresponder), the prevalence of which within each strain is strikingly similar to our previous observations in males. Within a given phenotype, rats of each strain performed similarly during operant testing. Ethanol intake during home cage drinking was unable to predict future operant phenotype. Relative to controls, Drinkers chronically exposed to ethanol liquid diet exhibited a significant, but transient, escalation in consummatory, but not appetitive, responding during acute withdrawal. Collectively, these data closely parallel many of our previous observations in males while also highlighting potential sex differences in drinking strategies following dependence. The presence of the Responder phenotype reinforces the importance of using direct measures of ethanol consumption. Our findings provide new insight into similarities and differences in operant ethanol self-administration between males and females and emphasize the importance of including females in future studies of ethanol drinking and dependence.

Epigenetic adaptation drives monocyte differentiation into microglia-like cells upon engraftment into the retina

Liu, J. — 2024-09-14

The identification of specific markers to distinguish resident microglia from infiltrating monocytes has been a long-standing challenge in neuroscience. Recently, proteins such as P2RY12, TMEM119, and FCRLS have been proposed as microglia-specific and are now widely used to define microglial populations in health and disease. The specificity of these markers was predicated on the assumption that circulating monocytes retain their distinct signatures after entering the central nervous system (CNS). Here, we challenge this paradigm. Using a combination of bone marrow chimeras, single-cell RNA sequencing, ATAC-seq, flow cytometry, and immunohistochemistry, we demonstrate that monocytes engrafting into the CNS acquire de novo expression of these established microglia markers. This phenotypic conversion is driven by profound epigenetic reprogramming, characterized by dynamic changes in chromatin accessibility at key gene loci, including P2ry12, Tmem119, and Aif1 (Iba1), and a shift in transcription factor binding motifs toward a microglial profile. We show this process occurs in the retina following injury and, remarkably, under physiological conditions in the brain and spinal cord, where blood-derived monocytes progressively contribute to the resident myeloid pool. Furthermore, engrafted monocytes downregulate canonical monocyte markers (Ly6C, CD45), eventually becoming indistinguishable from embryonic microglia based on conventional phenotyping. Our findings reveal that infiltrating monocytes undergo extensive epigenetic and transcriptional remodeling to adopt a microglia-like fate, challenging the specificity of current markers and necessitating a re-evaluation of the distinct roles of these two cell populations in CNS pathology. Significance StatementDistinguishing resident CNS microglia from infiltrating monocytes is fundamental to understanding neuro-inflammation. This study reveals that widely used "microglia-specific" markers are not exclusive, as monocytes entering the central nervous system are epigenetically reprogrammed to express them. This mimicry invalidates long-held assumptions about microglial identity and demonstrates that many cells previously identified as microglia may have a peripheral origin. Our work underscores the critical need for more reliable methods to differentiate these populations to accurately define their respective contributions to CNS health and disease.

A Statistically-Robust Model Of The Axomyelin Unit Under Normal Physiologic Conditions With Application To Disease States

Gow, A. — 2024-09-14

Despite tremendous progress in characterizing the myriad cellular structures in the nervous system, a full appreciation of the interdependent and intricate interactions between these structures is as yet unfulfilled. Indeed, few more so than the interaction between the myelin internode and its ensheathed axon. More than a half-century after the ultrastructural characterization of this axomyelin unit, we lack a reliable understanding of the physiological properties, the significance and consequence of pathobiological processes, and the means to gauge success or failure of interventions designed to mitigate disease. Herein, we highlight shortcomings in the most common statistical procedures used to characterize the axomyelin unit, with particular emphasis on the underlying principles of simple linear regression. These shortcomings lead to insensitive detection and/or ambiguous interpretation of normal physiology, disease mechanisms and remedial methodologies. To address these problems, we syndicate insights from early seminal myelin studies and use a statistical model of the axomyelin unit that is established in the accompanying article. Herein, we develop and demonstrate a statistically-robust analysis pipeline with which to examine and interpret axomyelin physiology and pathobiology in two disease states, experimental autoimmune encephalomyelitis and the rumpshaker mouse model of leukodystrophy. On a cautionary note, our pipeline is a relatively simple and streamlined approach that is not necessarily a panacea for all g ratio analyses. Rather, it approximates a minimum effort needed to elucidate departures from normal physiology and to determine if more comprehensive studies may lead to deeper insights.

Biofabrication of engineered tissues by 3D bioprinting of tissue specific high cell-density bioinks

Jeon, O. — 2024-09-15

Bioprinting of high cell-density bioinks is a promising technique for cellular condensation-based tissue engineering and regeneration medicine. However, it remains difficult to create precisely controlled complex structures and organization of tissues with high cell-density bioink-based bioprinting for tissue specific condensation. In this study, we present newly biofabricated tissues from directly assembled, tissue specific, high cell-density bioinks which have been three-dimensionally printed into a photocrosslinkable and biodegradable hydrogel microparticle supporting bath. Three types of tissue specific high cell-density bioinks have been prepared with individual stem cells or stem cell aggregates by incorporation of growth factor-loaded gelatin microparticles. The bioprinted tissue specific high cell-density bioinks in the photocrosslinked microgel supporting bath condense together and differentiate down tissue-specific lineages to form multi-phase tissues (e.g., osteochondral tissues). By changing the growth factors and cell types, these tissue specific high cell-density bioinks enable engineering of various functional tissues with controlled architecture and organization of cells.

A modular system for programming multistep activation of endogenous genes in stem cells

Puppala, A. K. — 2024-09-17

Although genomes encode instructions for mammalian cell differentiation with rich syntactic relationships, existing methods for genetically programming cells have modest capabilities for stepwise regulation of genes. Here, we developed a sequential genetic system that enables transcriptional activation of endogenous genes in a preprogrammed, stepwise manner. The system relies on the removal of an RNA polymerase III termination signal to induce both the transcriptional activation and the DNA endonuclease activities of a Cas9-VPR protein to effect stepwise progression through cascades of gene activation events. The efficiency of the cascading system enables a new dimension for cellular programming by allowing the manipulation of the sequential order of gene activation for directing the differentiation of human stem cells. One-Sentence SummaryDevelopment of a synthetic biology system for preprogrammed, stepwise activation of endogenous genes.

Protrusion force and cell-cell adhesion play a critical role in collective tumor cluster migration

Wang, H. — 2024-09-22

Collective migration refers to the coordinated movement of cells as a single unit during migration. While collective migration enhances invasive and metastatic potential in cancer, the mechanisms driving this behavior and regulating tumor migration plasticity remain poorly understood. This study provides a mechanistic model explaining the emergence of different modes of collective migration under hypoxia-induced secretome. We focus on the interplay between cellular protrusion force and cell-cell adhesion using collectively migrating three-dimensional microtumors as models with well-defined microenvironments. Large microtumors show directional migration due to intrinsic hypoxia, while small microtumors exhibit radial migration when exposed to hypoxic secretome. Here, we developed an in silico multi-scale microtumor model (MSMM) based on the cellular Potts model and implemented in CompuCell3D to elucidate underlying mechanisms. We identified distinct migration modes within specific regions of protrusion force and cell-cell adhesion parameter space and studied these modes using in vitro experimental microtumor models. We show that sufficient cellular protrusion force is crucial for radial and directional collective microtumor migration. Radial migration emerges when sufficient cellular protrusion force is generated, driving neighboring cells to move collectively in diverse directions. Within migrating tumors, strong cell-cell adhesion enhances the alignment of cell polarity, breaking the symmetric angular distribution of protrusion forces and leading to directional microtumor migration. The integrated results from the experimental and computational models provide fundamental insights into collective migration in response to different microenvironmental stimuli. Our computational and experimental models can adapt to various scenarios, providing valuable insights into cancer migration mechanisms. Statement of SignificanceWhile single-cell metastasis is well-studied, mechanisms of collective cluster migration are less understood. Significant challenges include the lack of a fundamental physics perspective on collective cluster migration mechanisms and suitable physiologically relevant three-dimensional (3D) in vitro models that can recapitulate collective cluster migration. In this article, we developed a computational model depicting microtumor migration behaviors. Collective cell migration, with varying correlation lengths, exhibits different migratory modes such as directional and radial migration. These modes are predicted by in silico models and confirmed using experimental microtumor models. Machine learning methods were exploited to identify migratory modes. Our computational and experimental models are flexible in various circumstances, offering insights into cancer migration mechanisms.

Cell Contractile Forces Drive Spatiotemporal Morphing in 4D Bioprinted Living Constructs

Cleveland, D. S. — 2024-09-24

Current 4D materials typically rely on external stimuli such as heat or light to accomplish changes in shape, limiting the biocompatibility of these materials. Here, a composite bioink consisting of oxidized and methacrylated alginate (OMA), methacrylated gelatin (GelMA), and gelatin microspheres is developed to accomplish free-standing 4D bioprinting of cell-laden structures driven by an internal stimulus: cell-contractile forces (CCF). 4D changes in shape are directed by forming bilayer constructs consisting of one cell-free and one cell-laden layer. Human mesenchymal stem cells (hMSCs) are encapsulated to demonstrate the ability to simultaneously induce changes in shape and chondrogenic differentiation. Finally, the capability to pattern each layer of the printed constructs is exhibited to obtain complex geometric changes, including bending around two separate, non-parallel axes. Bioprinting of such 4D constructs mediated by CCF empowers the formation of more complex constructs, contributing to a greater degree of in vitro biomimicry of biological 4D phenomena.

Dopamine activity encodes the changing valence of the same stimulus in conditioned taste aversion paradigms

Loh, M. — 2024-10-01

Mesolimbic dopamine encoding of non-contingent rewards and reward-predictive cues has been well established. Considerable debate remains over how mesolimbic dopamine responds to aversion and in the context of aversive conditioning. Inconsistencies may arise from the use of aversive stimuli that are transduced along different neural paths relative to reward or the conflation of responses to avoidance and aversion. Here, we made intraoral infusions of sucrose and measured how dopamine and behavioral responses varied to the changing valence of sucrose. Pairing intraoral sucrose with malaise via injection of lithium chloride (LiCl) caused the development of a conditioned taste aversion (CTA), which rendered the typically rewarding taste of sucrose aversive upon subsequent re-exposure. Following CTA formation, intraoral sucrose suppressed the activity of ventral tegmental area dopamine neurons (VTADA) and nucleus accumbens (NAc) dopamine release. This pattern of dopamine signaling after CTA is similar to intraoral infusions of innately aversive quinine and contrasts with that to sucrose when it was novel or not paired with LiCl. Dopamine responses were negatively correlated with behavioral reactivity to intraoral sucrose and predicted home cage sucrose preference. Further, dopamine responses scaled with the strength of the CTA, which was increased by repeated LiCl pairings and weakened through extinction. Thus, the findings demonstrate differential dopamine encoding of the same taste stimulus according to its valence, which is aligned to distinct behavioral responses.

Sociality shapes mitochondrial adaptations supporting hypoxia tolerance

Rossi, A. — 2024-10-02

Oxygen deprivation or hypoxia is poorly dealt with by most terrestrial species and often leads to permanent tissue damage and death. One prominent exception is the naked mole-rat (Heterocephalus glaber) which is remarkably adapted to withstand prolonged periods ([~]18 mins) of severe hypoxia, a trait likely driven by its crowded underground lifestyle. Other African mole-rat species are less social or entirely solitary like the Cape mole-rat (Georychus capensis). Here, we asked whether cellular and molecular adaptations to hypoxia map to social traits. We discovered that at the cellular level naked mole-rat fibroblasts survive >30 hours in 1% oxygen, while fibroblasts from terrestrial or non-social mole-rat species (human, mouse and Cape mole-rat) die rapidly under hypoxic conditions. We further show that naked mole-rat mitochondria have evolved morphological, functional and proteomic adaptations crucial for hypoxia resistance, remaining unaffected after long periods of severe hypoxia. We identify the mitochondrial protein Optic Atrophy 1 (OPA1) as a key player in naked mole-rat hypoxia resilience. Naked mole-rat mitochondria not only express more protective forms of OPA1, but also harbor a structurally unique isoform that likely protects cells from hypoxic damage. We show that evolutionary changes including the functionalization of a unique Opa1 exon support mitochondrial mediated cellular protection. Indeed, knockdown of OPA1 in naked mole-rat cells is sufficient to render them equally susceptible to hypoxia as human cells or cells from non-social African species. Our study demonstrates how molecular evolution drives unique adaptations that enable cells to achieve unprecedented resistance to hypoxic damage. We also show that molecular changes at the level of mitochondria are crucial in conferring hypoxia resistance. Our results thus chart a novel molecular path to understand how robust cellular hypoxia resistance can be achieved. Such knowledge may eventually inspire novel strategies to circumvent the consequences of hypoxic-damage in humans.

Increased CPT1a expression is a critical cardioprotective response to pathological stress that suppresses gene programs for remodeling and enables rescue by gene transfer

Carley, A. N. — 2024-10-02

Text AbstractO_ST_ABSBackgroundC_ST_ABSCarnitine palmitoyl transferase 1 (CPT1) is a rate-limiting enzyme for long chain fatty acid oxidation (FAO) in cardiac mitochondria. In adult hearts, CPT1b predominates, while CPT1a is co-expressed at lower levels. Pathological stress on the heart induces greater CPT1a expression, and this coincides with a reduction in FAO, yet the role of CPT1a in pathological cardiac remodeling is unknown. MethodsCPT1 isoform expression was assayed in myocardium of human heart failure (HF) patients with nonischemic cardiomyopathy (NICM) and a preclinical mouse model of heart failure. To explore the role of CPT1a upregulation in response to pathological stress, mice were subjected to afterload stress via transverse aortic constriction (TAC) or sham surgery (sham) with cardiac-specific CPT1a knockdown or cardiac-specific, AAV9-mediated CPT1a overexpression (AAV9.cTNTN.Cpt1a), versus empty virus or PBS infusions as controls. MiR370, known to suppress hepatic CPT1a, was assayed and overexpressed to determine if miR370 regulates cardiac CPT1a expression. ResultsCPT1a protein was elevated and miR370 reduced in myocardium of male and female NICM patients (204% vs. non-failing unused donor hearts), as well as in failing mouse hearts. AAV mediated miR370 overexpression in mouse hearts suppressed CPT1a expression and attenuated the response of CPT1a to TAC. Preventing CPT1a upregulation in response to TAC in cardiac specific CPT1a knockout mice (csCPT1a ko) exacerbated adverse remodeling, causing severe dysfunction and increased mortality. In contrast, CPT1a overexpression (2.8 fold), attenuated impaired ejection fraction (EF, by 54%) and fractional shortening (FS, 65%) vs. PBS-infused TAC hearts (p<0.05). Delivery of AAV9.cTnT.Cpt1a 4 wks after TAC surgery, led to significant rescue of EF and FS vs. animals receiving empty virus and mitigated the exacerbated dysfunction of csCPT1a ko hearts at 4 wks TAC. RNA-seq and reverse transcription-quantitative PCR revealed a novel function of CPT1a in suppressing hypertrophic, profibrotic and cell death gene programs in both sham and TAC hearts, irrespective of changes in FAO. ConclusionsThe effects of CPT1a in the heart extend beyond FAO and include a non-canonical regulation of cardiac gene programs. In addition to an animal model of HF, CPT1a upregulation occurs in NICM, and is a critical cardioprotective adaptation to pathological stress.

IgG autoantibodies in bullous pemphigoid directly induce a pathogenic MyD88-dependent pro-inflammatory response in keratinocytes:

Bao, L. — 2024-10-11

AbstractWhile autoantibodies in bullous pemphigoid (BP) are known to activate the innate immune response, their direct effect on keratinocytes, and the contribution of BP-IgG autoantibody-dependent keratinocyte responses to BP pathology is largely unknown. Herein, we performed multiplex immunoassays and bulk RNA-seq on primary keratinocytes treated with IgG from BP patients or controls. We identified a pro-inflammatory and proteolytic response with release of several cytokines (IL-6, IL-24, TGF-{beta}1), chemokines (CXCL16, CTACK, MIP-3{beta}, RANTES), C1s, DPP4, and MMP-9. We further validated this response using spatial transcriptomics and scRNA-seq of diseased and control skin. Blistering itself appeared to be major driver of this inflammatory response, with attached BP skin and spongiotic dermatitis revealing highly similar transcriptomes. Based on elevated levels of MyD88 and MyD88-dependent cytokines, we studied the impact of MyD88 deficiency in keratinocytes and demonstrated that MyD88 regulates BP-IgG-induced expression of IL-8, IL-24, and MMP-9. Induction of experimental BP in mice with Krt14-specific Myd88 knockout revealed significantly decreased disease severity with decreased serum levels of IL-1{beta}, IL-4, and IL-9 indicating the contributory role of keratinocyte-derived skin inflammation towards systemic response. Our work demonstrates the key contributions of keratinocyte and MyD88 dependent signaling in response to autoantibodies in BP. Key Messages-IgG antibodies from bullous pemphigoid (BP) patients induce significant upregulation of several inflammatory markers in keratinocytes including cytokines (IL-6, IL-24, TGF-{beta}1), chemokines (CXCL16, CTACK, MIP-3{beta}, RANTES), C1s, DPP4, and MMP9. Several of these markers, including IL-8, IL-24, and MMP9 are regulated by MyD88. -Spatial transcriptomics reveals that BP patient blistered skin demonstrated similar transcriptomic profiles to BP-IgG-treated keratinocytes. With attached skin demonstrating a comparable transcriptome to that seen in spongiotic dermatitis. -In a mouse BP model, keratinocyte-specific MyD88 deficiency results in decreased disease severity with a subsequent decrease in serum IL-1{beta}, IL-4, and IL-9 levels. Capsule summaryIgG from patients with bullous pemphigoid (BP) induces a pro-inflammatory response in keratinocytes, indicating their direct role in driving the inflammatory response in BP.

Catabolism of extracellular glutathione supplies amino acids to support tumor growth

Hecht, F. — 2024-10-11

Restricting amino acids from tumors is an emerging therapeutic strategy with significant promise. While typically considered an intracellular antioxidant with tumor-promoting capabilities, glutathione (GSH) is a tripeptide of cysteine, glutamate, and glycine that can be catabolized, yielding amino acids. The extent to which GSH-derived amino acids are essential to cancers is unclear. Here, we find that GSH catabolism promotes tumor growth. We show that depletion of intracellular GSH does not perturb tumor growth, and extracellular GSH is highly abundant in the tumor microenvironment, highlighting the potential importance of GSH outside of tumors. We find supplementation with GSH can rescue cancer cell survival and growth in cystine-deficient conditions, and this rescue is dependent on the catabolic activity of {gamma}-glutamyltransferases (GGTs). Finally, pharmacologic targeting of GGTs activity prevents the breakdown of circulating GSH, lowers tumor cysteine levels, and slows tumor growth. Our findings indicate a non-canonical role for GSH in supporting tumors by acting as a reservoir of amino acids. Depriving tumors of extracellular GSH or inhibiting its breakdown is potentially a therapeutically tractable approach for patients with cancer. Further, these findings change our view of GSH and how amino acids, including cysteine, are supplied to cells.

The genetic basis of replicated bullseye pattern reduction across the Trionum Complex

Yeo, M. T. S. — 2024-10-12

Angiosperm flowers exhibit a wide diversity of colorful motifs on their petals. Such patterns fulfill both biotic and abiotic functions, mediating plant-pollinator communication and providing protection against damaging UV rays or desiccation. These motifs are often evolutionary labile, varying in size, shape and hue between closely related species and constitute excellent systems to illuminate the evolutionary processes that generate morphological diversity or instead lead to the repetitive emergence of similar forms. Hibiscus trionum flowers have a prominent bullseye pattern combining a purple center contrasting against a white margin. H. trionum belongs to a small clade of Hibiscus known as the Trionum Complex that displays a range of petal patterns within and between species. Here, we integrate phylogenomic approaches, molecular techniques and genetic manipulation to solve species relationships across the Trionum Complex, identify key genes involved in the production of bullseye pigmentation, and reveal molecular events underpinning pattern variation during the evolution of the group. We find that repetitive bullseye reduction events primarily occur through independent modifications of a single genetic locus encoding BERRY1, an R2R3 MYB that regulates anthocyanin pigment production in petals. Moreover, we demonstrate that buff-tailed bumblebees (Bombus terrestris) discriminate against flowers with smaller bullseye sizes suggesting that a reduction in bullseye proportions potentially impacts plant fitness and contributes to trait distribution across the group. Collectively, our results demonstrate how repeated mutations in a single locus led to morphological variation in petal patterning, a trait that contributes to angiosperm reproductive isolation and speciation.

Amplification of olfactory transduction currents implements sparse stimulus encoding

Belonio, K. C. — 2024-10-12

Sensory systems must perform the dual and opposing tasks of being sensitive to weak stimuli while also maintaining information content in dense and variable sensory landscapes. This occurs in the olfactory system, where OSNs are highly sensitive to low concentrations of odors and maintain discriminability in complex odor environments. How olfactory sensory neurons (OSNs) maintain both sensitivity and sparsity is not well understood. Here, we investigated whether the calcium-activated chloride channel, TMEM16B, may support these dual roles in OSNs. We used multiphoton microscopy to image the stimulus-response density of OSNs in the olfactory epithelium. In TMEM16B knockout mice, we found that sensory representations were denser, and the magnitude of OSN responses was increased. Behaviorally, these changes in sensory representations were associated with an increased aversion to the odorant trimethylamine, which switches perceptual valence as its concentration increases, and a decreased efficiency of olfactory-guided navigation. Together, our results indicate that the calcium-activated chloride channel TMEM16B sparsens sensory representations in the peripheral olfactory system and contributes to efficient integrative olfactory-guided behaviors.

hu.MAP3.0: Atlas of human protein complexes by integration of > 25,000 proteomic experiments

Fischer, S. N. — 2024-10-15

Macromolecular protein complexes carry out most functions in the cell including essential functions required for cell survival. Unfortunately, we lack the subunit composition for all human protein complexes. To address this gap we integrated >25,000 mass spectrometry experiments using a machine learning approach to identify > 15,000 human protein complexes. We show our map of protein complexes is highly accurate and more comprehensive than previous maps, placing [~]75% of human proteins into their physical contexts. We globally characterize our complexes using protein co-variation data (ProteomeHD.2) and identify co-varying complexes suggesting common functional associations. Our map also generates testable functional hypotheses for 472 uncharacterized proteins which we support using AlphaFold modeling. Additionally, we use AlphaFold modeling to identify 511 mutually exclusive protein pairs in hu.MAP3.0 complexes suggesting complexes serve different functional roles depending on their subunit composition. We identify expression as the primary way cells and organisms relieve the conflict of mutually exclusive subunits. Finally, we import our complexes to EMBL-EBIs Complex Portal (https://www.ebi.ac.uk/complexportal/home) as well as provide complexes through our hu.MAP3.0 web interface (https://humap3.proteincomplexes.org/). We expect our resource to be highly impactful to the broader research community.

ZMYND11 Functions in Bimodal Regulation of Latent Genes and Brain-like Splicing to Safeguard Corticogenesis

Chang, X. — 2024-10-16

Despite the litany of pathogenic variants linked to neurodevelopmental disorders (NDD) including autism (ASD) and intellectual disability1,2, our understanding of the underlying mechanisms caused by risk genes remain unclear. Here, we leveraged a human pluripotent stem cell model to uncover the neurodevelopmental consequences of mutations in ZMYND11, a newly implicated risk gene3,4. ZMYND11, known for its tumor suppressor function, encodes a histone-reader that recognizes sites of transcriptional elongation and acts as a co-repressor5,6. Our findings reveal that ZMYND11-deficient cortical neural stem cells showed upregulation of latent developmental pathways, impairing progenitor and neuron production. In addition to its role on histones, ZMYND11 controls a brain-specific isoform switch involving the splicing regulator RBFOX2. Extending our findings to other chromatin-related ASD risk factors revealed similar developmental pathway activation and splicing dysregulation, partially rescuable through ZMYND11s regulatory functions.

Branched-chain amino acid catabolism promotes ovarian cancer cell proliferation via phosphorylation of mTOR

Lusk, H. J. — 2024-10-18

Ovarian cancer is the sixth leading cause of cancer-related mortality among individuals with ovaries, and high-grade serous ovarian cancer (HGSOC) is the most common and lethal subtype. Characterized by a distinct and aggressive metastatic pattern, HGSOC can originate in the fallopian tube with the transformation of fallopian tube epithelial (FTE) cells, which metastasize to the ovary and subsequently to the omentum and peritoneal cavity. The omentum is a privileged metastatic site, and the metabolic exchange underlying omental metastasis could provide enzyme or receptor targets to block spread. In this study, we adapted a mass spectrometry imaging (MSI) protocol to investigate spatial location of 3D cocultures of tumorigenic FTE cells when grown in proximity to murine omental explants as a model of early metastatic colonization. Our analysis revealed several altered metabolites in tumorigenic FTE/omentum cocultures, namely changes in branched-chain amino acids (BCAA), including valine. We quantified the heightened consumption of valine, other BCAAs, and other amino acid-derived metabolites in omental cocultures using LC-MS assays. Our analysis revealed that metabolite concentrations when monitored with MSI from cell culture media in living culture systems have notable considerations for how MSI data may produce signatures that induce ionization suppression. Supplementation with valine enhanced proliferation and mTOR signaling in tumorigenic FTE cells, suggesting the potential of BCAAs as a nutrient utilized by tumor cells during omental colonization and a possible target for metastasis. SIGNIFICANCEThis study uncovers altered amino acid metabolism, specifically increased BCAA catabolism, at the interface of ovarian cancer cells and omental tissue in a coculture model of HGSOC secondary metastasis. Enhanced BCAA catabolism may promote cancer cell proliferation through mTOR signaling, presenting potential therapeutic value. These findings deepen our understanding of HGSOC pathogenesis and the metastatic tumor microenvironment, offering insights for developing new treatment strategies.

Efficient cell-wide mapping of mitochondria in electron microscopic volumes using webKnossos

Jiang, Y. — 2024-10-21

Recent technical advances in volume electron microscopy (vEM) and artificial intelligence-assisted image processing have facilitated high throughput quantifications of cellular structures, such as mitochondria that are ubiquitous and morphologically diversified. A still often overlooked computational challenge is to assign cell identity to numerous mitochondrial instances, for which both mitochondrial and cell membrane contouring used to be required. Here, we present a vEM reconstruction procedure (called mito-SegEM) that utilizes virtual path-based annotation to assign automatically segmented mitochondrial instances at the cellular scale, therefore bypassing the requirement of membrane contouring. The embedded toolset in webKnossos (an open-source online annotation platform) is optimized for fast annotation, visualization, and proofreading of cellular organelle networks. We demonstrate broad applications of mito-SegEM on volumetric datasets from various tissues, including the brain, intestine, and testis, to achieve an accurate and efficient reconstruction of mitochondria in a use-dependent fashion.

Association of oropharyngeal cancer recurrence with tumor-intrinsic and immune-mediated sequelae of reduced genomic instability

Sannigrahi, M. K. — 2024-11-04

BackgroundLimited understanding of the biology predisposing certain human papillomavirus-related (HPV+) oropharyngeal squamous cell carcinomas (OPSCCs) to relapse impedes therapeutic personalization. We aimed to identify molecular traits that distinguish recurrence-prone tumors. Methods50 HPV+ OPSCCs that later recurred (cases) and 50 non-recurrent controls matched for stage, therapy, and smoking history were RNA-sequenced. Groups were compared by gene set enrichment analysis, and select differences were validated by immunohistochemistry. Features discriminating groups were scored in each tumor using gene set variation analysis, and scores were evaluated for recurrence prediction ability. ResultsCases downregulated pathways linked to anti-tumor immunity (FDR-adjusted p<.05) and contained fewer tumor-infiltrating lymphocytes (p<.001), including cytotoxic T-cells (p=.005). Cases also upregulated pathways related to cell division and other aspects of tumor progression. Upregulated and downregulated pathways were respectively used to define a tumor progression score (TPS) and immune suppression score (ISS) for each tumor. Correlation between TPS and ISS (r=.603, p<.001) was potentially explained by observed upregulation of DNA repair pathways in cases, which might enhance their progression directly and by limiting cytosolic DNA-induced inflammation. Accordingly, cases contained fewer double-strand breaks based on staining for phospho-RPA32 (p=.006) and {gamma}-H2AX (p=.005) and downregulated pro-inflammatory components of the cytoplasmic DNA sensing pathway. A combined score derived from TPS and ISS optimized recurrence prediction and stratified survival in a manner generalizable to three external cohorts. ConclusionsWe provide novel evidence that limiting genomic instability makes tumor-intrinsic and immune-mediated contributions to HPV+ OPSCC recurrence risk, opening opportunities to detect and target this treatment-resistant biology.

Small molecule modulator of neuronal lysosome positioning and function resolves Alzheimers Disease-linked pathologies in cultured human neurons

Snead, A. M. — 2024-11-05

Abnormal increase in axonal lysosome abundance is associated with multiple neurodegenerative diseases including Alzheimers disease. However, the underlying mechanisms and disease relevance are not fully understood. We have recently identified RH1115 as a small molecule modulator of the autophagy-lysosomal pathway that regulates lysosome positioning in neurons. This allowed us to manipulate neuronal lysosome distribution including in axons and interrogate its contribution to both optimal neuronal functioning and to disease pathology. We demonstrate that the small molecule not only rescues aberrant buildup of both axonal autophagic and lysosomal intermediates but also reduces secreted A{beta}42 levels in human iPSC-derived neurons lacking the lysosomal adaptor, JIP3. We thus demonstrate that restoring efficient axonal lysosome transport has an anti-amyloidogenic effect in human neurons and is a promising therapeutic strategy for Alzheimers disease. Furthermore, we show that the small molecule enhances neuronal lysosome degradation, requires the lysosomal adaptor JIP4 to rescue the axonal lysosome pathology in the JIP3 KO neurons and increases levels of the JIP4-interacting lysosomal membrane protein, TMEM55B. Lastly, treatment with the small molecule led to a striking rescue of locomotor defects in JIP3 KO zebrafish larvae. Thus, we have identified a small molecule which can be impactful in neurodegenerative diseases that have a lysosomal pathology and have determined its molecular targets in modulating axonal lysosome abundance.

Tardigrade Dsup: Interactions with DNA and protection of cells from oxidative stress

Ni, G. S. — 2024-11-08

The remarkable capability of Tardigrade to survive under extreme conditions has been partially attributed to Dsup, an intrinsically disordered, highly positively charged protein. Dsup has been shown to bind to DNA in vitro, a property that has been associated with the capability of Dsup to exhibit stress-protective effects when expressed in mammalian cells. However, DNA binding of Dsup has not been visualized in living cells and expression of Dsup in different cell types was associated with either protective or detrimental effects. In addition, the effect of Dsup expression has not been clearly demonstrated at the organism level. Here we combined molecular dynamics (MD) simulations and fluorescence lifetime imaging microscopy (FLIM)-Forster resonance energy transfer (FRET) to interrogate Dsup-DNA interactions and demonstrated Dsup binding to DNA in living mammalian cells. Furthermore, Dsup expression in both HEK293T cells and yeast enhanced cell survival in the presence of hydrogen peroxide, suggesting that the presence of Dsup allows both mammalian and yeast cells to better cope with oxidative stress conditions. This study provides a better understanding of the property and functional role of Dsup and lays a foundation to explore new approaches to enhance stress resistance.

Accumulation of Dietary and Non-Enzymatic Oxysterols in Preeclamptic Placentas: Insights into Cholesterol Dynamics in Pregnancy Complications

Zou, L. — 2024-11-14

Oxysterols--oxidized derivatives of cholesterol--play key roles in regulating lipid metabolism, oxidative stress, and immune signaling. Despite their biological importance, their presence and function in human placental tissue remain poorly understood, particularly in pregnancy complications such as preeclampsia. Preeclampsia is a hypertensive disorder of pregnancy associated with oxidative stress and altered lipid homeostasis. This study profiled both dietary and non-enzymatic oxysterols in third-trimester human placentas from healthy term pregnancies and those complicated with preeclampsia, delivered at term or preterm and term. Findings indicate significantly higher oxysterol levels in preeclamptic placentas, particularly for 7-ketcholesterol (7-Keto) and 5,6{beta}-cholestanetriol, (Triol) regardless of gestational age. 5{beta},6{beta}-epoxycholesterol (OCallaghan, Woods, & OBrien, 2001) was higher in term preeclampsia, while total cholesterol levels were significantly lower in preeclamptic placentas. Correlation analyses revealed distinct associations between oxysterols, cholesterol, and total fat content, indicating unique oxidative signatures in preeclamptic pregnancies. These findings contribute to a growing body of evidence suggesting that altered oxysterol profiles may reflect oxidative stress and placental dysfunction. While causality cannot be inferred, oxysterol profiling may hold potential as a complementary biomarker strategy for identifying pregnancies at risk of adverse outcomes.

Clickable PEG-norbornene microgels support suspension bioprinting and microvascular assembly

Zhang, I. W. — 2024-11-15

The development of perfusable and multiscale vascular networks remains one of the largest challenges in tissue engineering. As such, there is a need for the creation of customizable and facile methods to produce robustly vascularized constructs. In this study, secondarily crosslinkable (clickable) poly(ethylene glycol)-norbornene (PEGNB) microbeads were produced and evaluated for their ability to sequentially support suspension bioprinting and microvascular self-assembly towards the aim of engineering hierarchical vasculature. The clickable PEGNB microbead slurry exhibited mechanical behavior suitable for suspension bioprinting of sacrificial bioinks, could be UV crosslinked into a granular construct post-print, and withstood evacuation of the bioink and subsequent perfusion of the patterned void space. Endothelial and stromal cells co-embedded within jammed RGD-modified PEGNB microbead slurries assembled into capillary-scale vasculature after secondary crosslinking of the beads into granular constructs, with endothelial tubules forming within the interstitial space between microbeads and supported by the perivascular association of the stromal cells. Microvascular self-assembly was not impacted by printing sacrificial bioinks into the cell-laden microbead support bath before UV crosslinking. Collectively, these results demonstrate that clickable PEGNB microbeads are a versatile substrate for both suspension printing and microvascular culture and may be the foundation for a promising methodology to engineer hierarchical vasculature.

Design and synthesis of triazine-based hydrogel for combined targeted doxorubicin delivery and PI3Kinhibition

Mandal, S. — 2024-11-21

Melanoma, an aggressive skin cancer originating from melanocytes, presents substantial challenges due to its high metastatic potential and resistance to conventional therapies. Hydrogels, three-dimensional networks of hydrophilic polymers with high water-retention capacities, offer significant promise for controlled drug delivery applications. In this study, we report the synthesis and characterization of hydrogelators based on the triazine molecular scaffold, which self-assemble into fibrous networks conducive to hydrogel formation. Rheological analysis confirmed their hydrogelation properties, while microscopic techniques including FE-SEM and FEG-TEM provided insights into their morphological networks. The drug delivery capability of these hydrogelators was evaluated using doxorubicin, a widely employed anticancer agent, demonstrating enhanced biocompatibility and reduced side effects compared to free doxorubicin. Additionally, the hydrogelators exhibited inhibitory activity against phosphoinositide 3-kinase (PI3K), a key enzyme frequently mutated in cancer, and also involved in melanoma progression. The dual functionality of this delivery system - controlled drug release and PI3K inhibition - highlights the potential of triazine-based hydrogelators as innovative therapeutic platforms for melanoma treatment.

Platelet Factor 4 (PF4) Regulates Hematopoietic Stem Cell Aging

Zhang, S. — 2024-11-27

Hematopoietic stem cells (HSCs) responsible for blood cell production and their bone marrow regulatory niches undergo age-related changes, impacting immune responses and predisposing individuals to hematologic malignancies. Here, we show that the age-related alterations of the megakaryocytic niche and associated downregulation of Platelet Factor 4 (PF4) are pivotal mechanisms driving HSC aging. PF4-deficient mice display several phenotypes reminiscent of accelerated HSC aging, including lymphopenia, increased myeloid output, and DNA damage, mimicking physiologically aged HSCs. Remarkably, recombinant PF4 administration restored old HSCs to youthful functional phenotypes characterized by improved cell polarity, reduced DNA damage, enhanced in vivo reconstitution capacity, and balanced lineage output. Mechanistically, we identified LDLR and CXCR3 as the HSC receptors transmitting the PF4 signal, with double knockout mice showing exacerbated HSC aging phenotypes similar to PF4-deficient mice. Furthermore, human HSCs across various age groups also respond to the youthful PF4 signaling, highlighting its potential for rejuvenating aged hematopoietic systems. These findings pave the way for targeted therapies aimed at reversing age-related HSC decline with potential implications in the prevention or improvement of the course of age-related hematopoietic diseases. Key PointsO_LIAge-related attrition of the megakaryocytic niche and associated PF4 downregulation is a central mechanism in HSC aging. C_LIO_LIPF4 supplementation, acting on LDLR and CXCR3 receptors, rejuvenates the function of aged HSCs. C_LI

Structure of the SMYD2-PARP1 Complex Reveals Both Productive and Allosteric Modes of Peptide Binding

Zhang, Y. — 2024-12-04

Allosteric regulation allows proteins to dynamically respond to environmental cues by modulating activity at sites away from the catalytic center. Despite its importance, the SET-domain protein lysine methyltransferase superfamily has been understudied. Here, we present four crystal structures of SMYD2, a unique family member with a MYND domain. Our findings reveal a novel allosteric binding site with high conformational plasticity and promiscuity, capable of binding peptides, proteins, PEG, and small molecules. This site exhibits positive cooperativity with substrate binding, influencing catalytic activity. Mutations here significantly alter substrate affinity, changing the enzymes kinetic profile. Specificity studies show interaction with PARP1 but not histones, suggesting targeted regulation. Interestingly, this sites function remains unaffected by active site changes, indicating unidirectional mechanisms. Our discovery provides novel insights into SMYD2s biochemical regulation and lays the foundation for broader research on allosteric control in lysine methyltransferases. Given SMYD2s role in various cancers, this work opens exciting avenues for designing specific allosteric inhibitors with reduced off-target effects.

Mycobacterium tuberculosis CrgA Forms a Dimeric Structure with Its Transmembrane Domain Sandwiched between Cytoplasmic and Periplasmic β-Sheets, Enabling Multiple Interactions with Other Divisome Proteins

Shin, Y. — 2024-12-06

CrgA is a key transmembrane (TM) protein in the cell division process of Mycobacterium tuberculosis (Mtb), the pathogen responsible for tuberculosis. While many of the Mtb divisome proteins have been identified, their structures and interactions remain largely unknown. Previous studies of CrgA using oriented-sample solid-state NMR have defined the tilt and rotation of the TM helices, but the cytoplasmic and periplasmic domains and even the oligomeric state were uncharacterized. Here, combining oriented-sample and magic-angle spinning solid-state NMR spectra, we solved the full-length structure of CrgA. The structure features a dimer with a TM domain sandwiched between a cytoplasmic {beta}-sheet and a periplasmic {beta}-sheet. The {beta}-sheets stabilize dimerization, which in turn increases CrgAs ability to participate in multiple protein interactions. Within the membrane, CrgA binds FtsQ, CwsA, PbpA, FtsI, and MmPL3 via its TM helices; in the cytoplasm, Lys23 and Lys25 project outward from the {beta}-sheet to interact with acidic residues of FtsQ and FtsZ. The structural determination of CrgA thus provides significant insights into its roles in recruiting other divisome proteins and stabilizing their complexes for Mtb cell wall synthesis and polar growth.

Heparanase, a host gene that potently restricts retrovirus transcription

Waxman, B. — 2024-12-06

Heparanase (HPSE) is a heterodimeric {beta}-D-glucoronidase that is critical in mammalian cells for the enzymatic cleavage of membrane associated heparan sulfate moieties. Apart from its enzymatic function, HPSE has important non-enzymatic functions, which include transcriptional regulation, chromatin modification and modulation of various signaling pathways. Interestingly, while HPSE is an interferon stimulated gene, past reports have shown that it has proviral properties for many different viruses, including Herpes Simplex Virus 1, as it assists virus release from infected cells. Yet any antiviral functions associated with HPSE have not been described. Here we show that HPSE utilizes a hitherto unknown mechanism to restrict retroviruses, by targeting the step of proviral transcription. Moreover, we demonstrate that HPSE blocks transcription initiation by targeting the SP1 transcription factor. Finally, we illustrate that the antiretroviral effect of HPSE is independent of its enzymatic activity. This report describes a novel antiviral mechanism utilized by HPSE to inhibit retrovirus infection. ImportanceHeparanase (HPSE) has emerged as an important factor that has proviral functions for a number of viruses including herpes simplex virus and hepatitis C virus, by assisting in virus egress. However, HPSE is an interferon stimulated gene and thus is a part of the host antiviral defense. Nothing is known about the antiviral functions of HPSE. Here, we examine in depth the role of HPSE during retrovirus infection using two retroviruses, human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). In this report, we show that mouse, but not human, HPSE blocks retrovirus infection by targeting provirus transcription. HPSE sequesters the SP-1 transcription factor away from the proviral promoter thereby inhibiting transcription inhibition. In conclusion, our findings identify a novel antiviral function of HPSE and its potential role as an inhibitor of zoonotic transmission of retroviruses.

MatriCom: a scRNA-Seq data mining tool to infer ECM-ECM and cell-ECM communication systems

Lamba, R. — 2024-12-16

The ECM is a complex and dynamic meshwork of proteins that forms the framework of all multicellular organisms. Protein interactions within the ECM are critical to building and remodeling the ECM meshwork, while interactions between ECM proteins and cell surface receptors are essential for the initiation of signal transduction and the orchestration of cellular behaviors. Here, we report the development of MatriCom, a web application (https://matrinet.shinyapps.io/matricom) and a companion R package (https://github.com/Izzilab/MatriCom), devised to mine scRNA-Seq datasets and infer communications between ECM components and between different cell populations and the ECM. To impute interactions from expression data, MatriCom relies on a unique database, MatriComDB, that includes over 25,000 curated interactions involving matrisome components, with data on 80% of the [~]1,000 genes that compose the mammalian matrisome. MatriCom offers the option to query open-access datasets sourced from large sequencing efforts (Tabula Sapiens, The Human Protein Atlas, HuBMAP) or to process user-generated datasets. MatriCom is also tailored to account for the specific rules governing ECM protein interactions and offers options to customize the output through stringency filters. We illustrate the usability of MatriCom with the example of the human kidney matrisome communication network. Last, we demonstrate how the integration of 46 scRNA-Seq datasets led to the identification of both ubiquitous and tissue-specific ECM communication patterns. We envision that MatriCom will become a powerful resource to elucidate the roles of different cell populations in ECM-ECM and cell-ECM interactions and their dysregulations in the context of diseases such as cancer or fibrosis. ONE SENTENCE SUMMARYMatriCom is a web application devised to mine scRNA sequencing datasets to infer ECM-ECM and cell-ECM communication systems in the context of the diverse cell populations that constitute any tissue or organ.

Metformin inhibits nuclear egress of chromatin fragments in senescence and aging

Kumazawa, T. — 2024-12-17

Chronic inflammation is a hallmark of aging and contributes to many age-associated diseases. Metabolic intervention is a strategy to modulate inflammation. However, the connection between inflammation and metabolism during aging remains poorly understood. A mechanism driving chronic inflammation involves cytoplasmic chromatin fragments (CCFs), which appear in senescent cells and aged tissues, activating the cGAS-STING pathway. The size of the CCFs exceeds the capacity of the nuclear pore complex, raising the question of how chromatin fragments enter the cytoplasm. Here, we report that chromatin fragments exit the nucleus via nuclear egress, a membrane trafficking process at the nuclear envelope that shuttles large complexes from the nucleus to the cytoplasm. Inactivating critical nuclear egress ESCRT-III or Torsin proteins results in accumulation of chromatin fragments at the nuclear membrane, thereby impairing the activation of cGAS-STING and senescence-associated inflammation. Notably, nuclear egress of CCFs is inhibited by glucose limitation or metformin treatment. This is due to AMPK phosphorylation and autophagic degradation of the ESCRT-III component, ALIX. Metformin treatment in naturally aged mice downregulates ALIX protein and blocks cGAS activation and chronic inflammation in the small intestine. Together, our study defines a central mechanism linking nutrient sensing and chronic inflammation, two distinct hallmarks of aging, and suggests a new approach to suppress age-associated inflammation by targeting the nuclear egress of chromatin fragments.

Image Processing in the Acute to Chronic Pain Signatures (A2CPS) Project

Sadil, P. — 2024-12-20

The Acute to Chronic Pain Signatures (A2CPS) project is a large-scale, multi-site initiative aimed at identifying biomarkers and biosignatures that predict the transition from acute to chronic pain. The project is collecting multimodal, longitudinal data from over 2,500 individuals at risk for developing chronic pain after surgery. Here we describe the neuroimaging component of A2CPS, including the acquisition protocols, processing pipelines, and contents of the initial data release. The imaging protocol includes structural, diffusion, resting-state and task-based functional magnetic resonance imaging (MRI) data. Data are collected across multiple clinical sites using different scanner manufacturers, with attention to protocol harmonization and quality control. The processing pipeline integrates several established neuroimaging tools to extract potential biomarkers, including measures of brain structure, connectivity, and pain-related neural signatures. The first data release includes pre-surgical imaging data for 595 participants, with high quality ratings across modalities (98.7% of sMRI, 99.8% of dMRI, and 94.6% of fMRI images were rated as acceptable or better). Initial analyses demonstrate expected relationships between brain-derived measures and clinical variables, such as associations between brain age and psychological factors. This dataset represents a valuable resource for both pain research and neuroimaging methods development, with future releases planned to include additional participants and expanded analysis pipelines and processed data derivatives.

Recombinant Pichinde reporter virus as a safe and suitable surrogate for high-throughput antiviral screening against highly pathogenic arenaviruses

Sacramento, C. Q. — 2024-12-20

Several arenaviruses, such as the Old World (OW) Lassa virus (LASV) and the New World (NW) Junin virus (JUNV), can cause severe and lethal viral hemorrhagic fevers in humans. Currently, no vaccines or specific antiviral therapies are FDA-approved for treating arenavirus infections. One major challenge for the development of new therapeutic candidates against these highly pathogenic viruses is that they are BSL-3/4 pathogens that need to be handled in high biocontainment laboratories. In this work, a recombinant non-pathogenic New World arenavirus, Pichinde virus (rPICV), was used for the development of a high-throughput screening (HTS) assay in the BSL-2 laboratory for the screening and identification of small molecule inhibitors against arenaviruses. The rPICV is a replication-competent virus expressing the firefly luciferase reporter gene in the infected cells proportionally to the infection rate. rPICV infection was optimized for an automated HTS in 384-well format with robust Z' scores, high signal-to-background ratios, and low intrinsic variance. Screening an established library allowed for the identification of five top hit compounds, which included ribavirin, a known inhibitor of arenaviral RNA synthesis, showing good potency and selectivity in inhibiting rPICV replication. The antiviral activity of the top hit compounds was further validated against another recombinant arenavirus, the OW lymphocytic choriomeningitis virus (rLCMV) and against laboratory strains of LASV (Josiah) and JUNV (Romero). The use of rPICV in the HTS-based antiviral assay under BSL-2 condition has proven to be safe and suitable for the identification of broad-spectrum small molecule inhibitors against highly pathogenic arenaviruses.

Pro-cognitive reshaping of neuronal dynamics by a human CSF-based factor

Dos Santos, M. — 2024-12-26

Neuronal connection dysfunction is a convergent cause of cognitive deficits in mental disorders. Cognitive processes are finely regulated at the synaptic level by membrane proteins, some of which are shed and detectable in patients cerebrospinal fluid (CSF). However, whether these soluble synaptic proteins can harnessed as innovative pro-cognitive factors to treat brain disorders remains unclear. Here, we use quantitative proteomics to identify shed synaptic proteins dysregulated in the CSF of subjects with schizophrenia (SCZ), a mental disorder characterized by cognitive and synaptic dysfunction. The level of a yet uncharacterized soluble form of the voltage-gated calcium channel auxiliary subunit, 2{delta}-1, is robustly reduced in SCZ CSF. Remarkably, soluble 2{delta}-1 is convergently downregulated across several brain disorder CSF proteomes. We show that the brain releases soluble 2{delta}-1 in an activity-dependent manner, which can reorganize neuronal network dynamics by binding to synaptic targets and promoting inhibitory neuron plasticity. A single brain injection of a synthetic soluble 2{delta}-1 improved interneuron and cognitive deficits in a mutant mouse model of SCZ and cortical dysfunction. These findings underscore the potential of shed synaptic proteins as novel therapeutic agents capable of enhancing brain function in diverse brain disorders characterized by cognitive impairment.

Noncanonical PI(4,5)P2 coordinates lysosome positioning through cholesterol trafficking

Loughran, R. M. — 2025-01-03

In p53-deficient cancers, targeting cholesterol metabolism has emerged as a promising therapeutic approach, given that p53 loss dysregulates sterol regulatory element-binding protein 2 (SREBP-2) pathways, thereby enhancing cholesterol biosynthesis. While cholesterol synthesis inhibitors such as statins have shown initial success, their efficacy is often compromised by the development of acquired resistance. Consequently, new strategies are being explored to disrupt cholesterol homeostasis more comprehensively by inhibiting its synthesis and intracellular transport. In this study, we investigate a previously underexplored function of PI5P4Ks, which catalyzes the conversion of PI(5)P to PI(4,5)P2 at intracellular membranes. Our findings reveal that PI5P4Ks play a key role in facilitating lysosomal cholesterol transport, regulating lysosome positioning, and sustaining growth signaling via the mTOR pathway. While PI5P4Ks have previously been implicated in mTOR signaling and tumor proliferation in p53-deficient contexts, this work elucidates an upstream mechanism that unifies these earlier observations.

Star-Shaped Glatiramer Acetate Mitigates Pulmonary Dysfunction and Brain Neurodegeneration in a Murine Model of Cryptococcus-Associated IRIS

Anwar, S. — 2025-01-10

Cryptococcal-associated immune reconstitution inflammatory syndrome (C-IRIS) is a clinical worsening or new presentation of cryptococcal disease following the initiation of antiretroviral therapy. C-IRIS is primarily driven by an influx of pathological CD4+ T cells, which triggers a hyperinflammatory response. The murine model of C-IRIS is a way to study the disease in mice and understand how the immune system triggers life-threatening outcomes in patients. We previously developed a murine C-IRIS model and demonstrated that C-IRIS is triggered by pathological CD4+ T cells, particularly Th1 cells, in the brain, which triggers neurodegeneration and pulmonary dysfunction. Using this unique mouse model, we tested the therapeutic effect of a star-shaped glatiramer acetate (sGA), which is a more effective isomeric form than linear GA. Here, we observed that sGA suppresses Th1 differentiation in the lung tissues, reducing CD4+ T cell and Th1 cell count. It also reduced microglia populations in the brain. Together, these changes improved respiratory dysfunction caused by C-IRIS, lowered mortality rate, and reduced brain neurodegeneration. These findings suggest that sGA could be an effective therapeutic strategy for managing C-IRIS.

Jk DNA GAGA MOTIFS ARE REQUIRED FOR LOCAL NUCLEOSOME REMODELING AND Vk-Jk RECOMBINATION

Veselits, M. — 2025-01-23

Immunoreceptor gene recombination requires complementary 12 bp and 23 bp recombination signal sequences (RSSs). In addition, the RSSs that assemble the RAG proteins, recombination centers, must be accessible yet flanked by a 5 nucleosome decorated with H3K4me3. In Drosophila, DNA GAGA motifs play an important role in nucleosome positioning. Herein, we report that 5 to each functional Jk RSS is a DNA GAGA motif conserved across mammalian species. In mice, the GAGA motif 5 to Jk1 regulated local RSS accessibility and 5 nucleosome placement. Furthermore, it was required for Vk-Jk1 recombination. Murine Jk3 is nonfunctional, having mutations in both RSS and GAGA motifs. Restoring both GAGA and RSS motifs rescued Vk-Jk3 recombination. In contrast, restoring the RSS alone did not. Genome-wide, strong cryptic 23 RSSs were preferentially bound to nucleosomes. Furthermore, evolutionary selection against cRSS only occurred in the A Compartment of B lymphocytes, not embryonic stem cells. These data indicate that in developing B cells, nucleosome positioning both enables and restricts recombination to Jk. Furthermore, our data suggest an expanded definition of recombination center-associated RSSs to include a 5 GAGA sequence that dictates the local epigenetic state required for gene recombination. SummaryRecombination center assembly requires a specific epigenetic topology at recombination signal sequences. Herein, we report that conserved GAGA motifs 5 to each Jk segment are required for establishing this epigenetic topology and subsequent local gene recombination.

Measuring bridging forces in protein-DNA condensates

Ahlawat, V. — 2025-01-30

Protein-DNA condensates mediate transcription and regulate gene expression and DNA replication and repair. The intermolecular bridging forces stabilizing condensates have direct roles in these processes. Here we use optical tweezers to measure bridging forces. In the presence of protamine, a single condensate is observed on a 20.5-knt single-stranded DNA (ssDNA) tethered between two microbeads. Stretching produces force curves with a sawtooth pattern, suggesting that the condensate is dissembled by the sequential rupture of individual protamine-ssDNA bridges. The bridging forces are 11.3 {+/-} 4.6 pN, with unfolding lengths of 1.3 {+/-} 0.8 {micro}m for single bridges. In contrast, double-stranded DNA (dsDNA) forms protamine-bridged tangles that can withstand forces high enough ([~]55 pN) for strand separation. ssDNA tracks unpeeled at nicks on dsDNA by overstretching seed tangle formation upon retraction, but the initial condensates have a sufficient ssDNA-to-dsDNA ratio to appear liquid-like, as indicated by a sawtooth pattern in the subsequent stretching. The presence of dsDNA raises bridging forces to 34 {+/-} 8 pN, which revert to [~]10 pN upon adding external ssDNA. In line with these single-molecule results, protamine-dsDNA mixtures form solid-like aggregates and require the addition of ssDNA to become liquid droplets. Conversely, adding dsDNA slows the fusion of protamine-ssDNA droplets. This work demonstrates the first measurements of bridging forces and shows that the ssDNA-to-dsDNA ratio can tune their magnitude in protein-DNA condensates.

Cell-type Specific Ribosomal Tagging Allows for Simultaneous Multi-Tissue Translatomic Analysis

Eng, J. — 2025-02-06

In vivo transcriptomic analysis has advanced significantly with the development of single cell technologies. However, bulk RNA sequencing also continues to provide information on critical signaling pathways and cellular responses. The isolation of mRNA by polysome immunoprecipitation can identify genes undergoing active translation. Unfortunately, the inability to profile multiple cell types from the same sample remains a major downfall of the technique and limits broader analysis of the tissue microenvironment. In this study, we demonstrated the feasibility of immunoprecipitating polysome-associated mRNA from different cell types by strategically expressing differently tagged Rpl22 subunits. Using this technique, we isolated two distinct sets of high quality transcripts from intact B16F10 melanomas, endothelial cells and B16F10 tumors cells, for further molecular analysis.

Suppressed macrophage responses to quorum-sensing-active Streptococcus pyogenes occurs at the level of the nucleus

Feldstein, S. F. — 2025-02-08

Streptococcus pyogenes, or Group A Streptococus (GAS), a significant human pathogen, employs quorum sensing (QS) systems to coordinate its behavior and genetic regulation in order to enhance survival. Our previous research established that one such QS system, the Rgg2/3 system, can suppress macrophage NF{kappa}B activity and production of pro-inflammatory cytokines. Yet, the scope of suppression and the mechanism by which it occurs remains unknown. In this study, we used transcriptomic and phosphoproteomic approaches to address these unanswered questions. We found QS-ON GAS broadly suppressed most inflammatory transcriptional pathways including those of NF{kappa}B, type I and type II interferon responses, and intracellular stress responses. Yet, we found no alternative transcriptional programs were activated after QS-ON GAS infection. Additionally, phosphoproteomics showed no disruption in typical inflammatory pathways such as those related to NF{kappa}B and MAPK activation, which was confirmed by western blotting and translocation assays. Instead, the proteomic data highlighted a potential role for epigenetic mechanisms of inflammatory regulation. To determine if epigenetic regulation was involved in QS-mediated immunomodulation, DNA methylation was measured and studies were performed inhibiting various histone and chromatin modifiers. These studies also showed no dijerence between QS-ON compared with QS-OFF infected macrophages. These findings expand our understanding of QS-mediated suppression and of GAS virulence strategies that appear to employ unusual methods of restricting inflammation. Uncovering this mechanism will ojer invaluable insight into GAS, itself, as well as understudied immunological pathways. ImportanceStreptococcus pyogenes is a ubiquitous pathogen that causes over 600 million infections every year and 500 thousand to 1 million fatalities. While in developed countries it is generally known to cause mild conditions such as pharyngitis, it can also manifest as severe infections such as necrotizing fasciitis, septic arthritis, and lead to post-infectious sequelae including rheumatic heart disease and glomerulonephritis. Elucidating new mechanisms of virulence in this organism, including how it evades and suppresses immune responses can be critical in understanding its pathogenicity, epidemiology, and identification of novel treatment avenues in this era of multi-drug-resistant bacteria. In this study, we characterize the broad spectrum by which GAS modulates the host innate immune response and begin to uncover host pathways that bacteria can use or inhibit for its survival.

Perception and neural representation of intermittent odor stimuli in mice

Boero, L. E. — 2025-02-13

Odor cues in nature are sparse and highly fluctuating due to turbulent transport. To investigate how animals perceive these intermittent cues, we developed a behavioral task in which head-restrained mice made binary decisions based on the total number of discrete odor pulses presented stochastically over several seconds. Mice readily learned this task, and their performance was well-described by widely used decision models. Logistic regression of binary choices against the timing of odor pulses within the respiratory cycle revealed that mice placed higher perceptual weight to stimuli arriving during inhalation than exhalation, a phase dependency that strongly correlated with the magnitude of responses in olfactory sensory neurons. The population response of anterior piriform cortex (APCx) neurons to odor pulses was also modulated by respiration phase, although individual neurons displayed varying levels of phase-dependence. Single APCx neurons responded stochastically and transiently to odor pulses, leading to a representation that carries signatures of sensory evidence, but not its accumulation. Our study reveals that mice can integrate intermittent odor signals across dozens of breaths, but respiratory modulation of sensory inputs imposes limits on information acquisition that cortical circuits cannot overcome to improve behavior.

Herbaria provide a valuable resource for obtaining informative mRNA

Tyszka, A. S. — 2025-02-17

While DNA has built the framework for molecular insights from museum collections, the utility of archival RNA remains largely unexplored. Likely a consequence of the known instability of RNA relative to DNA, this has effectively precluded the use of herbaria for transcriptomics. Here, we challenge the assumption that herbaria cannot be used for transcriptomics by assembling transcriptomes from RNA extracted from herbarium specimens. Through systematic comparison of transcriptomes from fresh-collected, silica-dried, and archival specimens, we demonstrate the suitability of herbarium-derived RNA for transcriptomics. We show the practical applicability of archival mRNA by functionally validating a plant immune receptor synthesized from a specimen collected in 1956. These results contradict the community consensus regarding archival RNA and open the door to subsequent transcriptomic explorations of rare and extinct plant species. Our findings highlight the importance of preserving and utilizing the diversity embedded within herbarium collections.

Oral Cavity Serves as Long-Term COVID-19 Reservoir with Increased Periodontal and Viral Disease Risk

Schwartz, J. — 2025-02-19

BackgroundSARS-CoV-2 infection can lead to long-term health problems affecting multiple body systems termed long COVID. Currently, limited information exists about long-term oral health manifestations in COVID-19 patients with limited healthcare access. MethodsWe conducted a sequential, cross-sectional study (December 2020-March 2024) to assess how racial/ethnic differences (Black/Hispanic vs White/Asian) and health disparities affect oral and non-oral long COVID symptoms and their relationship with COVID-19 vaccination. We retrospectively reviewed patients oral health record from University of Illinois Chicago dental clinics before vaccination (December 2020; N=1150; Covid+/- N=575/group) and after vaccination (December 2021; N=592; Covid+/- N=292/group). Participants were recruited in two separate prospective groups of COVID-19 positive subjects (February-April 2021; pre-vaccination: N=158; January-March 2024; post-vaccination: N=171), we examined clinical indicators of oral (periodontal and salivary glands) and non-oral (neurologic) sequalae 3-6 months after initial exposure. We measured viral S protein by flow cytometry and quantified inflammatory markers, viral entry receptors, and oral viral load to correlate molecular, and cellular changes in COVID-19 positive subjects before and after vaccination. ResultsOur results identified racial disparity indicating oral associated post-acute sequelae (PASC) primarily manifested as periodontal (gum) disease (COVID-19 positive: 73.1{+/-}18.9% vs COVID-19 negative: 33.1{+/-}14.3%) and correlated with higher rates of dry mouth (57.5%), taste disturbance (47%), and smell loss (20%). Vaccination reduced oral PASC in COVID-19 positive subjects; however, periodontal disease indicators persisted compared to the COVID-19 negative group. Notably, 3-6 months post-infection, while SARS-CoV-2 Spike (S) transcript was rarely detected in saliva ([~]6%), its protein was commonly detected ([~]70%) in the COVID-19 positive subjects indicating incomplete viral clearance. This correlates with significantly higher salivary expression of viral entry receptors (ACE2, and TRMPSS2), and inflammatory mediators (IL-6, IL-8 and MMP-8), in COVID-19 positive subjects. This finding was further supported by higher prevalence of other oral viruses including Epstein-Barr Virus (70.5%), Herpes Simplex Virus (8.1%), and Human Papillomavirus (17.5%) in COVID-19 positive subjects. InterpretationCOVID-19 history significantly correlates with severe oral health complications in predominantly Black communities, while vaccination reduced but did not eliminate these issues. The oral cavity serves as a long-term viral reservoir, and periodontal inflammation with increased oral viral presence in COVID-positive patients may increase susceptibility to oral and non-oral viral diseases and identify risk for long COVID.

Cholesterol efflux protein, ABCA1, supports anti-cancer functions of myeloid immune cells

Bendre, S. V. — 2025-02-19

Although immune therapy has seen significant advances, the majority of breast and other solid tumors do not respond or quickly develop de novo resistance. One factor driving resistance is highly immune suppressive myeloid cells (MCs) such as macrophages. Previous work has established clinical links between cholesterol and cancer outcome, and that MC function can be regulated through disruption in cholesterol metabolism. Thus, we screened for proteins that were expressed in MCs, involved in cholesterol homeostasis and whose expression was associated with survival; we identify the cholesterol efflux protein ABCA1. Preclinical studies revealed that ABCA1 activity resulted in increased anti-cancer functions of macrophages: enhanced tumor infiltration, decreased angiogenic potential, reduced efferocytosis, and improved support of CD8+ T cell activity. Mechanistically, different AKT isoforms are involved, through both PI3K dependent and independent mechanisms. Assessment of human blood and breast tumors revealed correlations between ABCA1 in macrophages and angiogenic potential, VEGFA, and CD8 T cell abundance and activity, highlighting the clinical relevance of our findings. The culmination of the effects of ABCA1 on MC function were demonstrated through increased tumor growth and metastasis in mice with MC specific knockout of ABCA1. Therefore, modulating ABCA1 activity within MCs may represent a novel approach to immune therapy.

Murine Leukemia Virus GlycoGag Antagonizes SERINC5 via ER-phagy Receptor RETREG1

Ahmad, I. — 2025-03-06

Serine incorporator 5 (SERINC5) is a host restriction factor that targets certain enveloped viruses, including human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). It integrates into the viral envelope from the cell surface, inhibiting viral entry. SERINC5 is transported to the cell surface via polyubiquitination, while a single K130R mutation retains it in the cytoplasm. Both HIV-1 Nef and MLV glycoGag proteins antagonize SERINC5 by reducing its expression in producer cells. Here, we report that MLV glycoGag employs selective autophagy to downregulate SERINC5, demonstrating a more potent mechanism for decreasing its cell surface expression. Although glycoGag is a type II integral membrane protein, it primarily localizes to the cytoplasm and undergoes rapid proteasomal degradation. Employing the K130R mutant, we show that Nef, primarily associated with the plasma membrane, downregulates SERINC5 only after it has trafficked to the cell surface, whereas glycoGag can reduce its expression before reaching the plasma membrane while still in the cytoplasm. Nonetheless, an interaction with SERINC5 stabilizes and recruits glycoGag to the plasma membrane, enabling it to downregulate SERINC5 from the cell surface. Through affinity-purified mass spectrometry analysis combined with CRISPR/Cas9 knockouts, we find that glycoGags activity depends on reticulophagy regulator 1 (RETREG1), an ER-phagy receptor. Further knockout experiments of critical autophagy genes demonstrate that glycoGag downregulates cytoplasmic SERINC5 via micro-ER-phagy. These findings provide crucial new insights into the ongoing arms race between retroviruses and SERINC5 during infection. AUTHOR SUMMARYHIV-1 Nef and MLV glycoGag are unrelated viral proteins, yet both counteract the same host restriction factor, SERINC5, to facilitate productive infection. In this study, we report a novel pathway through which glycoGag downregulates SERINC5. We demonstrate that while Nef downregulates SERINC5 only after it has trafficked to the cell surface, glycoGag can directly downregulate SERINC5 in the cytoplasm before it reaches the plasma membrane. Furthermore, we show that this pathway is mediated by the ER-phagy receptor RETREG1, which targets SERINC5 for degradation via micro-ER-phagy. This mechanism provides a more effective means of blocking SERINC5 antiviral activity. These findings reveal that retroviruses have evolved different strategies to antagonize SERINC5, highlighting the critical role of SERINC5 in restricting retroviral infections.

In situ cell-only bioprinting of patterned prevascular tissue into bioprinted high-density stem cell-laden microgel bioinks for vascularized bone tissue regeneration

Jeon, O. — 2025-03-18

Recently, microgels have been widely used in three-dimensional (3D) bioprinting as both supporting baths and bioinks. As a bioink, microgels have several unique properties, such as shear-thinning and self-healing behaviors with tunable mechanics, making them useful in 3D bioprinting. While cell encapsulated microgels offer many advantages in 3D bioprinting, they also have some limitations. It is still challenging to produce large quantities of cell encapsulated microgels with consistent quality and properties due to processes that are often complex and time-consuming. In this study, stem cell encapsulated, photocrosslinkable, shear-thinning and self-healing alginate microgel (SSAM) bioinks have been successfully fabricated via simple mixing of an oxidized and methacrylated alginate solution with suspended stem cells and a supersaturated calcium sulfate slurry solution through a custom-made spiral mixing unit. The SSAM bioinks can be bioprinted into complex 3D structures with both high resolution and shape fidelity due to their shear-thinning and self-healing properties. The 3D bioprinted SSAM bioinks can then serve as a supporting bath for the creation of prevascular network patterns using an individual cell-only prevasculogenic bioink within the 3D printed constructs. The prevascular network patterned 3D bioprinted constructs can be further stabilized by secondary photocrosslinking of the SSAMs, which enables long-term culture of the printed constructs for functional vascularized osteogenic tissue formation by differentiation of the bioprinted cells. The SSAM bioinks and individual cell-only printing technique enable in situ bioprinting of prevascularized tissue constructs in a mouse calvarial bone defect, achieving mechanical stability and ensuring the in situ bioprinted constructs remain within the defect.

Creation and validation of LIMON - Longitudinal Individual Microbial Omics Networks

Alvernaz, S. A. — 2025-03-19

Microbial communities are dynamic structures that continually adapt to their surrounding environment. Such communities play pivotal roles in countless ecosystems from environmental to human health. Perturbations of these community structures have been implicated in disease processes such as Crohns disease and cancer. Disturbances to existing ecosystems often occur over time, making it essential to have robust methods for detecting longitudinal alterations in microbial interactions as they develop. Existing methods for identifying temporal microbial community alterations have focused on abundance alterations in individual taxa, rather than relationships between the taxa, known as microbial interactions. Identifying these interactions overtime provides a fuller understanding of how the microbial ecosystem changes as a whole. To fill this gap, we have developed a pipeline that handles the complicated nature of repeated compositional count data, LIMON - Longitudinal Individual Microbial Omics Networks. This novel statistical approach addresses key challenges of modeling temporal and microbial data including overdispersion, zero-inflated count data, compositionality, repeated measure design sample covariates over time, and identification of individualized or sample specific networks. This approach allows users to denoise covariate effects from their data, return networks per time point, identify interaction changes between each time point, and return individual networks and network characteristics per sample/time point. In doing so, LIMON provides a platform to identify the relationship between network interactions and sample features of interest over time. Here we show LIMON, in simulation studies, can accurately remove covariate effects, render sample specific networks, and better recover underlying network edges from covariate confounded data. Analysis of a longitudinal infant microbiome and diet dataset illustrates LIMONs novel utility to identify key microbial interactions related to diet type across time. AUTHOR SUMMARYMicrobes (bacteria, fungi etc.) are integral components of many ecosystems, from the environment to the human body, where they can shift between healthy and disease states. Microbes do not exist alone but in rich diverse communities. Yet, many current methods used to study microbe alterations in disease focus on changes in individual microbes rather than how the entire community adapts. To better understand how microbial communities shift, we developed an open-source tool called LIMON, which allows users study how these relationships shift over time. By leveraging robust statistical techniques, LIMON can account for the complexities of the data, such as covariates and differences between individual samples. This approach helps us uncover important patterns in how microbes interact in various conditions. In this example, we applied LIMON to data from infants who were fed three different diets during the first year of life and identify specific microbial interactions related to diet that change overtime. This work broadens the scope for exploring microbial ecosystem dynamics in health and nature, offering a more comprehensive perspective vs traditional method used.

Correlated Segments of Intrinsically Disordered Proteins as Drivers of Homotypic Phase Separation

Zhou, H.-X. — 2025-04-07

Many studies have suggested that amino acid composition, not their positions along the sequence, is the determinant of phase separation of intrinsically disordered proteins (IDPs). In particular, aromatic amino acids and Arg have been identified as major drivers. Here I underscore the importance of the positions of amino acids along the sequence in phase separation. Specifically, clusters of interaction-prone amino acids, including Trp and Arg, along the sequence form correlated segments, and these correlated segments, rather than individual residues, drive the phase separation of many IDPs. Correlated segments manifest themselves as stretches of residues that span major peaks in the backbone 15N NMR transverse relaxation rates and can be predicted by a sequence-based method called SeqDYN (https://zhougroup-uic.github.io/SeqDYNidp/). Inter-chain interactions between individual residues may be too transient, but those between correlated segments involve multiple residues can provide the strengths required for phase separation. Indeed, sequence motifs revealed by NMR and other techniques as important for phase separation frequently map to SeqDYN-predicted correlated segments. These include residues G624-R626, G638-R640, and R660-Q666 of CAPRIN1, residues R21-G30 of LAF-1, and residues Q9-P21 of FUS. SeqDYN presents a sequence-based method for identifying motifs that drive phase separation of IDPs.

TWIST1 mediated transcriptional activation of SPON2 drives colorectal peritoneal metastasis through activation of cancer-associated fibroblast signaling network

Zhou, Z. — 2025-04-17

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer-related mortality in the United States. Peritoneal metastasis (PM), a malignant dissemination within the peritoneal cavity, affects approximately 20% of CRC patients and accounts for 25-35% of stage IV cases. CRC PM is associated with dismal outcomes, with a median overall survival of only 16 months on systemic chemotherapy and an almost 0% five-year survival rate, largely due to frequent treatment resistance and limited therapeutic options. Despite advances in understanding CRC metastasis, the molecular mechanisms driving CRC PM remain poorly defined. CRC heterogeneity is classified into four Consensus Molecular Subtypes (CMS1-4), with CRC PM tumors predominantly exhibiting the CMS4 signature--characterized by stromal enrichment, high mesenchymal gene expression, and enhanced cellular plasticity--features linked to aggressive disease progression and resistance to standard chemotherapy. In this study, we identify TWIST1, a basic helix-loop-helix transcription factor, as significantly upregulated in CRC PM. We establish TWIST1-SPON2 as a novel transcriptional axis driving CRC PM tumorigenesis, mediating tumor-stroma interactions between tumor epithelium and cancer-associated fibroblasts (CAFs). Additionally, we identify SPP1, secreted by CAFs, as an upstream regulator of the TWIST1-SPON2 cascade via AKT activation in tumor cells. This newly defined SPP1-TWIST1-SPON2 signaling circuit plays a pivotal role in shaping the tumor microenvironment and promoting CRC PM progression. The findings establish the SPP1-TWIST1-SPON2 axis as a potential biomarker and a promising therapeutic target in CRC PM. Keyword: Colorectal cancer, peritoneal metastasis, epithelial-mesenchymal transition, cancer-associated fibroblast

Dpp Scaling is Not the Key to Robust Wing Patterning in Drosophila

Ghag, B. A. — 2025-04-24

Individuals within a population can vary dramatically in body and trait size in response to environmental factors such as developmental nutrition, temperature, and oxygen level, a phenomenon called phenotypic plasticity. Despite this variation, morphological pattern across the body and within traits are largely maintained, an example of developmental robustness. Consequently, the same trait can display both plastic and robust phenotypes. A fundamental yet unanswered question in developmental biology is: how do organs achieve plasticity in size while maintaining the robustness of pattern? This question is often answered using the French flag model of cell fate specification1-4. The model argues that cells receive positional information from a gradient of signaling molecules (morphogens) that diffuse from a single location in undifferentiated tissue. Specifically, the model argues that if the gradient is dynamically scale invariant, such that the gradient adjusts proportionally to the size of the tissue as it grows, then pattern is maintained regardless of the tissue size. However, while dynamic scale invariance has been demonstrated in several systems5,6, and explicitly2 or implicitly7,8 used to explain the robustness of pattern when tissue size varies due to environmental factors, this hypothesis has not yet been tested. We used the developing Drosophila wing as a model to test this hypothesis, under environmental conditions that reduce (low developmental oxygen level) and increase (low developmental temperature) adult wing size. We found that scale invariance of morphogen gradients is not observed under non-standard environmental conditions and is therefore not an explanation for robust patterning in the Drosophila wing with environmental variation in wing size.

DIRseq: a method for predicting drug-interacting residues of intrinsically disordered proteins from sequences

MacAinsh, M. — 2025-05-02

Intrinsically disordered proteins (IDPs) are now well-recognized as drug targets. Identifying drug-interacting residues is valuable for both optimizing compounds and elucidating the mechanism of action. Currently, NMR chemical shift perturbation and all-atom molecular dynamics (MD) simulations are the primary tools for this purpose. Here we present DIRseq, a fast method for predicting drug-interacting residues from the amino-acid sequence. All residues contribute to the propensity of a particular residue to be drug-interacting; the contributing factor of each residue has an amplitude that is determined by its amino-acid type and attenuates with increasing sequence distance from the particular residue. DIRseq predictions match well with drug-interacting residues identified by NMR chemical shift perturbation and other methods, including residues L22WK24 and Q52WFT55 in the tumor suppressor protein p53. These successes augur well for deciphering the sequence code for IDP-drug binding. DIRseq is available as a web server at https://zhougroup-uic.github.io/DIRseq/ and has many applications, such as virtual screening against IDPs and designing IDP fragments for in-depth NMR and MD studies.

Harmonized Protocol for Subfield Segmentation in the Hippocampal Body on High-Resolution in vivo MRI from the Hippocampal Subfields Group (HSG)

Daugherty, A. M. — 2025-05-04

Hippocampal subfields differentially develop and age, and they vary in vulnerability to neurodegenerative diseases. Innovation in high-resolution imaging has accelerated clinical research on human hippocampal subfields, but substantial differences in segmentation protocols impede comparisons of results across laboratories. The Hippocampal Subfields Group (HSG) is an international organization seeking to address this issue by developing a histologically-valid, reliable, and freely available segmentation protocol for high-resolution T2-weighted 3 tesla MRI (http://www.hippocampalsubfields.com). Here, we report the first portion of the protocol focused on subfields in the hippocampal body; protocols for the head and tail are in development. The body protocol includes definitions of the internal boundaries between subiculum, Cornu Ammonis (CA) 1-3 subfields, and dentate gyrus, in addition to the external boundaries of the hippocampus apart from surrounding white matter and cerebrospinal fluid. The segmentation protocol is based on a novel histological reference data set labeled by multiple expert neuroanatomists. With broad participation of the research community, we voted on the segmentation protocol via online survey, which included detailed protocol information, feasibility testing, demonstration videos, example segmentations, and labeled histology. All boundary definitions were rated as having high clarity and reached consensus agreement by Delphi procedure. The harmonized body protocol yielded high inter- and intra-rater reliability. In the present paper we report the procedures to develop and test the protocol, as well as the detailed procedures for manual segmentation using the harmonized protocol. The harmonized protocol will significantly facilitate cross-study comparisons and provide increased insight into the structure and function of hippocampal subfields across the lifespan and in neurodegenerative diseases.

Fibronectin Composition and Transglutaminase 2 Cross-linking Cooperatively Regulate Ovarian Cancer Cell Adhesion in ECM-Mimetic Constructs

Yang, N. — 2025-05-16

The extracellular matrix (ECM) plays a crucial role in tumor progression. Here, we analyzed collagen I and cellular fibronectin (cFN) in normal omentum and metastatic omentum from high-grade serous ovarian cancer (HGSOC). The levels of both proteins were significantly elevated and collagen I fibers were significantly thicker in HGSOC metastases. Moreover, the ECM cross-linking enzyme transglutaminase 2 (TG2) was increased in omental metastases, where it is enzymatically active in the extracellular environment. This information was used to develop ECM constructs recapitulating these key changes, alone and in combination, to investigate their impact on HGSOC cell adhesion. To our knowledge, this is the first report using TG2 as a cross-linking agent to generate constructs from multiple ECM components. Low levels of HGSOC cell adhesion were observed on colIagen-only (coll) gels, while inclusion of cFN or plasma fibronection (pFN) increased cell adhesion. TG2-mediated cross-linking of colI/cFN hydrogels promoted HGSOC cell adhesion, while cross-linking of coll/pFN had no effect. Cell adhesion was dependent on ligand identity and fiber diameter. When fiber thickness was held constant, the inclusion of cFN led to greater HGSOC cell adhesion relative to pFN or coll, due to interactions of {beta}1 integrins with the EDA and RGD domains of cFN. Meanwhile, when gel composition was held constant, HGSOC cell adhesion increased as fiber thickness was increased through modifications to gelation temperature. Combined, our results demonstrate how ECM changes associated with omental metastasis can support tumor progression and provide insights into methods to tailor biomaterials to support cell adhesion.

Endothelial c-IAP2 Loss Amplifies P2X7 Receptor-Driven Inflammation and Worsens Infection-Associated Pulmonary Hypertension

Villarreal, E. S. — 2025-05-27

Schistosomiasis-associated Pulmonary Hypertension (Sch-PH) is the most common form of group I PH worldwide. Recently, data revealed that the preclinical animal model of Sch-PH exhibited gut and lung microbiome dysbiosis linked to significant endothelial dysfunction and microvascular apoptosis, but the role of pro/anti-apoptosis sensors, such as the inhibitor of apoptosis protein 2 (c-IAP2) and purinergic receptor P2X7 (P2X7R), remained unclear. Using a novel Cdh5cre-ERT2;cIAP1-/-;cIAP2fl/fl animal model, this study investigated the contribution of endothelial c-IAP2 in this process, revealing P2X7R overexpression as a putative target in the onset of Sch-PH. Pharmacologically, inhibition of P2X7R function confirmed its role in promoting lung endothelial death and disease progression. Moreover, data suggest that microbiome-associated metabolic alterations in Sch-PH seem linked to microvascular endothelial apoptosis driven by ATP/P2X7R overactivation and suppressed c-IAP2 expression. Indeed, genetic ablation of endothelial c-IAP2 expression was sufficient to induce PH-like features in mice, with echocardiography indicating a higher pulmonary acceleration time (PAT), PAT/pulmonary ejection time (PET), and right ventricular free wall thickness after IP/IV-Egg challenge compared to controls. These findings suggest a significant contribution of lung endothelial P2X7R activation and c-IAP2 suppression to Sch-PH pathology, highlighting them as promising novel therapeutic targets for this life-threatening illness.

Sedative choice alters Klebsiella pneumoniae lung pathogenesis and dissemination

Mains, D. — 2025-05-27

Klebsiella pneumoniae make up 85% of carbapenem-resistant Enterobacteriaceae (CRE), bacteria that have become an urgent threat to public health. K. pneumoniae is largely transmitted in healthcare settings, where inpatients and outpatients are often anesthetized with the widely-used anesthetic induction agent propofol. Recent evidence obtained from rodent infection models indicates that propofol exposure can dramatically increase host susceptibility to microbial infections. Given that intensive care patients who are at a greater risk for K. pneumoniae lung infections are often given propofol during their hospitalization, we investigated the outcome of K. pneumoniae infections in mice briefly sedated with either propofol or ketamine/xylazine as control. Propofol-sedated mice experienced more rapid dissemination from the lungs to secondary sites of infection and their lungs exhibited more severe pathology. Based on these observations, we investigated bacterial factors involved in infection and dissemination in mice with propofol or ketamine/xylazine sedation using a high throughput insertion sequencing (INSeq) approach. We identified numerous novel potential virulence factors together with previously identified gene products, confirming the validity of our screen. We further characterized a mutant lacking the phospholipid retrograde trafficking chaperone MlaC and found that the degree of mutant attenuation was dependent upon sedation method. These results highlight the importance of sedative choice when studying hospital-acquired microbial infections and suggest that sedation can influence outcome of K. pneumoniae infection and dissemination in animal models. IMPORTANCEHost sedation by either propofol or ketamine exposure differentially impacted the severity of K. pneumoniae lung infection following intranasal inoculation of mice. While propofol-sedated mice exhibited increased lung pathology, some bacterial mutants, such as those lacking the MlaC gene product associated with the maintenance of inner and outer membrane lipid asymmetry, exhibited more severe attenuation following propofol sedation versus ketamine/xylazine. Given the dominating use of propofol in health care settings for the induction and maintenance of anesthesia, procedural sedation, and sedation for intensive care patients, this work provides important perspective as to how the choice of an anesthetic agent may impact the outcome of healthcare-associated infections.

Tolerance to Lung Infection in TWIK2 K+ Efflux Mediated Macrophage Trained Immunity

Thompson, J. — 2025-05-28

Lung macrophages such as alveolar macrophages (AM{varphi}) are essential for innate immune function in the lungs. It is now apparent that macrophages can be trained to become better at attacking infections. Although trained immunity is thought to result from metabolic and epigenetic reprogramming, the underlying mechanisms remain unclear. Here, we report that macrophages can be trained by extracellular ATP, which is ubiquitously released during inflammation. ATP ligates the canonical Purinergic Receptor 2 subtype X7 receptor (P2X7) to mediate endosomal Two-pore domain Weak Inwardly rectifying K+ channel 2 (TWIK2) translocation into the plasma membrane (PM). This endows the cells to transit to a ready state for microbial killing in two directions: first, K+ efflux via PM-TWIK2 induces NLRP3 inflammasome activation, which further activates metabolic pathways; second, upon bacterial phagocytosis, PM-TWIK2 internalizes into phagosome membrane with proper topological orientation, where TWIK2 mediates K+ influx into phagosomes to control pH and ionic strength favoring bacterial killing. Therefore, the enhanced association of TWIK2 in phagosomal and plasma membranes signaled by danger-associated molecular patterns (DAMPs), such as ATP, mediates trained immunity in macrophages and enhances the microbiocidal activity.

A systematic investigation of endothelial cell behavior under hydrostatic pressure

Venturini, G. — 2025-05-29

Biomechanical stimuli are critical in regulating cell behavior and phenotype across various tissues and organs, particularly within the cardiovascular system. Endothelial cells, which line blood vessels, are continuously subjected to forces generated by the pulsatile nature of blood flow, including shear stress, strain, and hydrostatic pressure (HP). Among these stimuli, HP remains the least explored, primarily due to the technical challenges of incorporating it into conventional cell culture systems. However, HP significantly influences key biological processes, such as cell differentiation, migration, proliferation, and apoptosis. To facilitate the introduction of HP in vitro, we have previously developed an automated, high-throughput platform compatible with standard 96-well plates capable of delivering up to 12 independent pressure conditions. In this study, we applied this setup to investigate the effects of a wide range of static pressure conditions on the viability, morphology, and cytoskeleton adaptation of Human Umbilical Vein Endothelial Cells (HUVECs).

GAN-Enhanced Machine Learning and Metabolic Modeling Identify Reprogramming in Pancreatic Cancer

Razmpour, T. — 2025-06-03

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the deadliest forms of cancer and presents a significant clinical challenge due to poor prognosis and limited treatment options. In this study, we developed a novel framework integrating genome-scale metabolic modeling (GSM) with machine learning to identify metabolic biomarkers and vulnerabilities in PDAC. We addressed the inherent class imbalance in cancer datasets by generating synthetic healthy samples using a Wasserstein Generative Adversarial Network with Gradient Penalty (WGAN-GP), implementing a three-step biological filtration process to ensure their validity. Our approach achieved 94.83% accuracy in distinguishing between healthy and cancerous metabolic states. Systems-level analysis revealed three key dysregulated pathways: heparan sulfate degradation, O-glycan metabolism, and heme degradation. We identified impaired lysosomal degradation of heparan sulfate proteoglycans as a potential contributor to PDAC pathogenesis, providing a mechanistic explanation for the previously observed association between lysosomal storage disorders and pancreatic cancer. Additionally, we found that nervonic acid transport (MAR00336) was the most discriminative reaction between healthy and cancerous states, with gene-level analysis highlighting FABPs, SLC27As, ACSLs, and ACSBGs as key molecular drivers of metabolic reprogramming in PDAC. Overall, our multi-level approach connected genetic drivers to functional metabolic consequences, revealing coordinated upregulation of fatty acid transport and activation processes. These findings enhance our understanding of PDAC metabolism and present potential therapeutic targets, demonstrating the value of integrated computational approaches in cancer research.

In vivo investigation of STN1 downregulation in melanoma formation in adult mice following UV irradiation

Knowles, S. — 2025-06-07

Genome instability is a major force driving tumorigenesis. The ssDNA-binding protein complex CTC1-STN1-TEN1 (CST) plays a pivotal role in maintaining genome stability by countering replication stress, modulating DNA damage repair, and maintaining telomere integrity. Despite its well-documented role in genome maintenance, the involvement of CST in skin cancer development has yet to be investigated. We recently found that CST localizes at stalled DNA replication sites after UV exposure and may suppress the unwanted repriming activity, suggesting a potential role of CST in suppressing genome instability caused by UV damage. In this study, we first analyzed CST expression and alterations in cutaneous melanoma database and found that the CST genes are frequently altered in cutaneous melanoma and their expression is significantly downregulated in melanoma samples compared to normal tissues. We then generated a conditional knockout (cKO) mouse model with STN1 deficiency specifically in melanocytes to investigate its role in skin cancer formation. Upon chronic exposure to UV irradiation, STN1-deficient mice exhibit no obvious difference in melanoma incidence compared to control littermates, suggesting that STN1 downregulation in mature melanocytes has no significant effect on UV-induced skin cancer development in lab mice.

Local optogenetic control of genome editing and tumorigenesis in vivo using wireless implantable optoelectronics

Bansal, T. — 2025-06-14

Precise spatial regulation of site-specific DNA recombination (SSR) in vivo remains a challenge due to limited tunability of current platforms. Here, we present an optogenetic approach that overcome these limitations by employing engineered light-regulated recombinase E-LightR-Cre and tunable wireless implantable optoelectronic devices. E-LightR-Cre meets the key criteria for spatial regulation of SSR in vivo, showing no detectable activity in the dark, while demonstrating robust activation upon blue-light illumination. To achieve local E-LightR-Cre activation in murine lungs, we developed wireless, fully-implantable optoelectronic devices enabling focal illumination with no discernible organ damage. By modulating illumination intensity and duration, we can control the size of the activated area. Local expression of oncogenic KRas-G12D in a photoactivated subpopulation of cells in vitro revealed rapid reprogramming of the mutant expressing cells and their non-activated neighbors. Light-guided activation of E-LightR-Cre in mouse lungs resulted in focal expression of a reporter gene and allowed us to induce local formation of oncogenic lesions in vivo.

A Multimodal Adaptive Optical Microscope For In Vivo Imaging from Molecules to Organisms

Fu, T.-M. — 2025-06-16

Understanding biological systems requires observing features and processes across vast spatial and temporal scales, spanning nanometers to centimeters and milliseconds to days, often using multiple imaging modalities within complex native microenvironments. Yet, achieving this comprehensive view is challenging because microscopes optimized for specific tasks typically lack versatility due to inherent optical and sample handling trade-offs, and frequently suffer performance degradation from sample-induced optical aberrations in multicellular contexts. Here, we present MOSAIC, a reconfigurable microscope that integrates multiple advanced imaging techniques including light-sheet, label-free, super-resolution, and multi-photon, all equipped with adaptive optics. MOSAIC enables non-invasive imaging of subcellular dynamics in both cultured cells and live multicellular organisms, nanoscale mapping of molecular architectures across millimeter-scale expanded tissues, and structural/functional neural imaging within live mice. MOSAIC facilitates correlative studies across biological scales within the same specimen, providing an integrated platform for broad biological investigation.

Going green: Recycling transcriptomes to infer evolutionary relationships, gene duplication, gene tree conflict, and patterns of molecular evolution in the Apocynaceae

Arreguin, S. — 2025-06-25

Background and AimsThe flowering plant family Apocynaceae exhibits diverse adaptations with biological and pharmaceutical significance, many of which have been studied with RNA-seq. However, despite the available transcriptomic data, no focused phylotranscriptomic study has been conducted to characterize the patterns of molecular evolution in this group. In this study, we leverage a dataset composed predominately of publicly available transcriptomes to infer relationships within Apocynaceae and explore the molecular processes that have shaped their divergences. MethodsWe extracted nuclear, chloroplast, and mitochondrial genes from 47 publicly available and one newly sequenced transcriptome to assemble and infer species relationships across Apocynaceae. Leveraging the gene-rich nuclear phylotranscriptomic data, we inferred molecular dates and investigated the complex history of gene tree conflict, molecular rate shifts, and gene duplications. To investigate the genomic basis of adaptations, we analyzed the inferred 14,838 gene duplications at the base of Apocynaceae for shared functional enrichment of genes related to evolutionary innovations. Key ResultsThe Apocynaceae topology inferred from our phylotranscriptomic analysis is highly concordant with the current consensus. Notably, a genome-wide acceleration in molecular rate subtends the Ceropegieae tribe. We observed that a decreased time between divergences is associated with a higher rate of gene tree conflict, a pattern especially prevalent across the Apocynaceaes historically recalcitrant backbone relationships. Furthermore, gene duplications may underlie evolutionary innovations, such as immunity-related gene expansion in the genus Asclepias and duplications associated with trichome modifications in the epiphytic Hoya. Finally, we discuss the contentious history of whole-genome duplication (WGD) within the Apocynaceae and emphasize the need for further investigation into the placement of WGDs. ConclusionsRepurposing transcriptomes is a powerful means of accumulating data for novel insights in plant evolution, especially during uncertain funding and cases where budget restrictions exist. Leveraging this gene-dense dataset, we obtained novel insights into the molecular evolution of Apocynaceae and identified areas for future investigations.

Smaller stepping thresholds in older adults might be related to reduced ability to suppress conflicting sensory information

Carey, H. D. — 2025-06-25

Aging leads to alterations in the sensorimotor system and balance control but it is not well understood how changes in sensorimotor function affect how people respond to postural disturbances. Elucidating the relationships between balance control and sensorimotor function is crucial for developing effective rehabilitations. Here, we compared the kinematic responses to platform translations and rotations during standing in 10 young and 30 older adults and explored relationships between sensorimotor function and balance responses. We found that older adults were less able to withstand perturbations without stepping, not because their non-stepping strategies were less effective but because they chose to step at smaller deviations of the extrapolated center of mass. Older adults performed worse than young adults on measures of sensory and motor function but lower stepping thresholds were associated with susceptibility to unreliable visual information and not with reduced sensory acuity or reduced strength. Poor sensory reweighting may contribute to and combine with age-related cognitive rigidity, leading to a higher priority on safer strategies. Older adults may resort to stepping, even if a step is not necessary, rather than rely on potentially inaccurate sensory signals to inform a corrective response. Our results provide initial evidence that sensory reweighting could be a potential target for fall prevention methods. NEW & NOTEWORTHYThe relationship between age-related changes in sensorimotor function and postural control is poorly understood. Here, we did a comprehensive assessment of sensorimotor function and reactive standing balance. We found that healthy older adults chose safer strategies, i.e. they step at smaller disturbances, than young adults. Although we found many differences in sensorimotor function, only a reduced ability to suppress conflicting sensory information was related to the use of a safer strategy.

A 4D Bio-Kirigami Strategy for Engineering Complex Tissue Curvatures

Gasvoda, K. L. — 2025-07-03

AbstractFour-dimensional (4D) systems offer a promising approach for generating sophisticated dynamic structures that mimic native tissue architectures. Among those, Kirigami strategies enable precise, localized control over morphing behaviors, yet their application in 4D tissue engineering remains unexplored. Here, we present a bio-Kirigami system that facilitates the creation of dynamic, transformable structures through spatially patterned hydrogels with distinct deformation modes dictated by encoded swelling differentials. The system consists of two biomaterial components: (1) a photocrosslinked hydrogel framework with controlled degradation and swelling behavior that drives the shape transformation and (2) a rigid support hydrogel frame. By leveraging photolithographic patterning, complex structures with continuously evolving configurations were achieved through preprogrammed deformations. Bio- Kirigami hydrogels encapsulating stem cells developed into tissue-like constructs with sophisticated configurations when cultured in tissue-specific environment. Notably, the engineered tissue constructs kept their shape integrity after excision from the outer support, demonstrating a robust platform for achieving intricate tissue curvatures.

Thin Filament Interaction and Ca2+-Desensitization Effect of the C-Terminal End Peptide of Cardiac Troponin T Where Loss of Function Mutations Cause Hypertrophic Cardiomyopathy

Li, Q. — 2025-07-10

BackgroundTroponin T (TnT) is the tropomyosin (Tm)-binding subunit of troponin with a central role in regulating cardiac muscle contractility. The recently identified Tm-binding site 3 in the highly conserved C-terminal end segment of TnT has a troponin I (TnI)-like inhibitory function. Conformational modulation by proteolytic removal of the N-terminal variable region of cardiac TnT (cTnT-ND) in adaptation to inotropy-afterload mismatch enhances the function of Tm-binding site 3 to adjust ventricular contractile kinetics and sustain stroke volume. Mutations in this segment of cTnT cause overactivation of cardiac muscle and hypertrophic cardiomyopathy (HCM). MethodsThe C-terminal end 14 amino acid peptide of cTnT (cTnT-C14) was analyzed for interactions with Tm, F-actin, and F-actin-Tm thin filament using localized surface plasmon resonance (LSPR). Wild-type and HCM mutant cTnT-C14 peptides were used to treat skinned cardiac muscle strips from wild-type and cTnT-ND transgenic mice to assess the effect on Ca{superscript 2}- activation of contraction. ResultscTnT-C14 peptide showed saturable binding to Tm and F-actin in LSPR with physiological affinity, which is significantly impaired by HCM mutation R278C, K280N or R286C. Treatment of wild-type mouse cardiac muscle strips with cTnT-C14 peptide produced a Ca2+- desensitization effect on myofilament activation, which was not seen with cTnT-ND mouse cardiac muscle strips implicating a prior utilization of the same mechanism. The HCM mutant peptides lost this function. ConclusionsThe findings demonstrate the function of the cTnT C-terminal end segment underlying the pathophysiology of the HCM mutations. Its preserved functionality in the form of free peptide presents a drug candidate for the treatment of heart failure.

Change-resistance distinguishes the representational geometries of human spatial memory and mouse CA1 in deformed environments

Green, D. M. — 2025-07-14

Prior work has highlighted qualitative similarities between the neural instantiations of cognitive maps in rodents and memory-guided navigation in humans, suggesting a conservation of representational structure across species. Yet evidence of cross-species differences in neural coding continues to mount. Our ability to reconcile these similarities and differences has been inherently limited by the qualitative nature of our cross-species comparisons. To overcome this limitation, here we combine recent technical and theoretical advances to characterize the representational geometry of human spatial memory during a diverse set of environmental deformations and compare this geometry to that of mouse CA1. Across three untethered immersive virtual reality experiments (n > 100 participants per experiment), we find that deformations induce compounding local distortions in human spatial memory. These distortions yield a representational geometry which closely resembles a change-resistant version of that of mouse hippocampal CA1 during analogous deformations. The geometries of mouse CA1 subpopulations with higher firing rates, spatial tuning stability, and spatial tuning specificity all better resembled that of human spatial memory. The precision, but not accuracy, of human spatial memory also modulated cross-species resemblance. The local impact of deformations scaled up when humans navigated a larger environment, preserving representational geometry and cross-species resemblance. Neither geometry nor cross-species resemblance depended on the human visual advantage during retrieval. Together, these results establish a common cross-species resemblance in the representational geometry of mouse CA1 and human spatial memory during environmental deformations, with a notable difference in the resistance to change between these assays.

Conformations and sequence determinants in the lipid binding of an adhesive peptide derived from Vibrio cholerae biofilm

Huang, X. — 2025-07-15

Surface adhesion is critical to the survival of pathogenic bacteria both in natural niches and during infections, often via forming matrix-embedded communities called biofilms. We previously identified a 57-amino acid peptide (Bap1-57aa) as a key contributor to biofilm adhesion of the pandemic pathogen Vibrio cholerae to various surfaces including lipid membranes. Here, we combine biophysical, computational, and genetic approaches to elucidate the molecular mechanism. A central aromatic-rich motif anchors the peptide to lipid bilayers while peripheral pseudo repeats enhance binding through avidity. Surprisingly, the core motif undergoes a lipid-induced conformational transition into a {beta}-hairpin, enabling robust membrane insertion. Moreover, the biofilm-derived peptide, conserved in several other Vibrio species, can adhere to model host surfaces and is sensitive to membrane curvature. Our results provide molecular insight into biofilm adhesion and may lead to new strategies for targeted biofilm removal and the design of bioinspired underwater adhesives. TeaserA short peptide from Vibrio cholerae binds lipids using a unique {beta}-hairpin motif and contributes to host colonization.

Comparative analysis of naked mole-rat thermogenesis and its potential to maintain euthermia in response to cold

Mikhalchenko, A. — 2025-07-17

The naked mole-rat (NMR) is a subterranean rodent known for its unique thermal biology, exceptional longevity and resistance to cancer and hypoxia. However, its thermal biology remains controversial, with various reports describing NMRs as poikilotherms, heterotherms, mesotherms or partial homeotherms. Here, we investigated whether the thermogenic potential of NMR brown adipose tissue and its UCP1 differ from those in mice and whether the lack of thermal insulation causes extreme changes in NMR body temperature upon cold exposure. Through longitudinal molecular, thermal, metabolic, and behavioral measurements, we found that NMRs initiated non-shivering thermogenesis and elevated body temperature but could not sustain it due to excessive heat loss and limits to substrate availability. Our results suggest that NMRs represent a unique thermoregulatory category that doesnt fit neatly into traditional classifications. In vitro and in vivo experiments showed that the NMR UCP1 is functional and can be activated and inhibited as expected for most other mammals. We further demonstrated that artificial insulation can partially restore thermoregulatory capabilities in NMRs. This study employs an advanced methodology to characterize the thermal biology of NMRs and helps resolve a long-standing controversy in the field. Significance StatementThis study provides critical insights into the thermal biology of naked mole-rats (NMRs), resolving long-standing controversies regarding their thermoregulatory strategies. We highlight the unique adaptations and limitations of NMR physiology by demonstrating that NMRs possess functional non-shivering thermogenesis and UCP1 but fail to maintain homeothermy due to excessive heat loss. Our findings suggest that artificial insulation can partially restore their thermoregulatory capabilities, offering a new perspective on the evolutionary and ecological significance of fur loss in NMRs. This research advances our understanding of mammalian thermal biology and presents an updated model for NMRs, bridging gaps between previous conflicting reports.

Stromal LOX/FAK/beta catenin pathway locks mammary fibroblasts into a tumor-promoting myCAF state

Gandhi, A. — 2025-07-17

BackgroundCancer-associated fibroblasts (CAFs) sustain tumor progression, yet the soluble cues that maintain their myofibroblast (myCAF) state are poorly defined. Transforming growth factor beta (TGF-{beta}) is a canonical CAF activator. This study aims to identify TGF-{beta}-induced secreted mediators that reinforce the myCAF phenotype in breast cancer and map the downstream signaling cascade. Methods and ResultsSecretome profiling of primary patient-derived myCAFs and human mammary fibroblasts (HMF3s) engineered to over-express TGF-{beta}1 revealed 20 extracellular-matrix remodelers shared exclusively by both activated states; lysyl oxidase (LOX) was the top-ranked hit. LOX knockdown abrogated TGF-{beta}-driven -smooth-muscle actin (-SMA) induction, collagen-gel contraction and migration in HMF3s, and reduced constitutive -SMA and {beta}-catenin in myCAFs. Mechanistically, TGF-{beta} upregulated LOX, which activated focal-adhesion kinase (FAK), leading to p38 MAPK- and Akt-mediated Ser9 phosphorylation (inactivation) of GSK3{beta} and consequent {beta}-catenin stabilization. In HCC1806-luciferase orthotopic xenografts, CAFs accelerated tumor growth, whereas LOX-deficient CAFs lost this pro-tumoral effect. ConclusionLOX is a pivotal autocrine effector of TGF-{beta} that locks breast CAFs into a pro-tumoral myCAF state through a LOX/FAK/GSK3{beta}/{beta}-catenin axis. Targeting stromal LOX may disrupt CAF activation and curb breast cancer progression.

MyeliMetric: A Python-Based Toolbox for Standardized G-ratio Analysis of Axon-Myelin Integrity

Ahmad, I. — 2025-07-18

The g-ratio, defined as the ratio of an axons diameter to the total fiber diameter (axon plus myelin), is a key metric for assessing myelin integrity and axonal conduction velocity in both the central and peripheral nervous systems. Deviations from the physiological range often signal underlying pathology. Despite its diagnostic importance, there is currently no standardized, open-source tool for g-ratio analysis from post-segmented electron microscopy images. To address this gap, we developed MyeliMetric, a Python-based, user-friendly toolbox that streamlines g-ratio data preprocessing and integrates biologically informed validation, requiring minimal statistical expertise to operate without introducing common analytical errors. It is built on the principle that g-ratios exhibit relative consistency across varying axon diameters in healthy conditions. To rigorously assess this relationship, MyeliMetric implements a binning strategy that groups axons into biologically relevant diameter cohorts, enabling the detection of size-dependent deviations in g-ratio distributions. This approach addresses common limitations in conventional analyses, including insufficient sampling, pseudo-replication, and artifacts such as misleading regression slopes. Validation using both synthetic and published datasets from rodent models of demyelination demonstrated the tools accuracy, reproducibility, and biological relevance. Synthetic data yielded expected outcomes, and in experimental models, MyeliMetric reliably detected reductions in myelin thickness through g-ratio shifts while minimizing artifacts, thereby providing biologically meaningful insights. It is available on GitHub: https://github.com/Intakhar-Ahmad/NeuroMyelin-G-Ratio-Analysis-Toolkit

Fast calculation of small-angle scattering profiles of dense protein solutions modeled at the all-atom level

Qin, S. — 2025-07-18

The small-angle scattering profile of a dense protein solution contains rich information about interprotein interactions, but extracting this information has been extremely challenging. In previous studies, we developed a fast Fourier transform (FFT)-based method, FMAPB2, to compute protein pair interactions at the all-atom level. Here, we use FMAPB2 to precompute pair interaction energies and then use the latter to drive simulations of dense protein solutions. The resulting MAEPPI (many-protein atomistic energy from pre-computed pair interaction) enables simulations of atomistic proteins at the speed of Lennard-Jones particles. On snapshots sampled from the simulations, we directly calculate the small-angle scattering profile. The results reveal artifacts generated by widely used spherical models and support a significant dimer population of bovine serum albumin at high concentrations. Our approach promises to modernize the prediction and interpretation of small-angle scattering profiles of dense protein solutions.

A novel interoceptive subfornical organ to infralimbic cortex circuit relays airway inflammation effects on fear extinction

Allgire, E. — 2025-07-18

There is growing interest in the impact of internal body states on the brain and behavior. The detrimental effects of chronic lung inflammation on mental health are well recognized, however, underlying mechanisms are not known. Here, using a murine model of allergic asthma we report compromised fear extinction in mice with severe but not mild airway inflammation (AI); an effect abolished by anti-interleukin-17A (IL-17A) antibodies. Investigation of innate immune cells, microglia as-well-as transcriptomic signatures in the subfornical organ (SFO), a brain interoceptive node lacking a traditional blood-brain-barrier, revealed significant alterations in severe AI mice. IL-17 Receptor A (IL-17RA) was expressed in SFO microglia and upregulated in severe AI mice. Notably, ablation of microglial IL-17RA improved fear extinction in severe AI mice. Furthermore, we identified direct SFO projections to the infralimbic (IL) cortex, a key area regulating extinction. Importantly, chemogenetic inhibition of the SFO-IL circuit led to improved fear extinction in severe AI mice. Collectively, we report a unique body-to-brain interoceptive mechanism engaging the SFO and an SFO-to-IL circuit, through which airway inflammatory mediators compromise fear extinction. Beyond asthma, our findings are relevant to other pulmonary pathologies (e.g. bacterial pneumonia, ARDS, COVID-19) highlighting a risk for cortical dysfunction and fear pathologies such as PTSD.

SOX2 utilizes FOXA1 as a heteromeric transcriptional partner to drive proliferation in therapy-resistant prostate cancer

Phoenix, J. T. — 2025-07-19

Treatment options and diagnostic outlook for men with advanced, therapy resistant prostate cancer (PCa) are extremely poor; this is primarily due to the common lack of durable response to androgen receptor (AR) targeted therapies and phenotypic transdifferentiation into a particularly lethal subtype known as neuroendocrine prostate cancer (NEPC). In this study, we mechanistically determine that SOX2 (a transcription factor originally repressed by AR) physically binds and acts in a concerted manner with FOXA1 (a key AR pioneering cofactor) to regulate a subset of genes which promote cell cycle progression, and lineage plasticity in AR-refractory prostate cancers. Our findings assert the SOX2/FOXA1 interaction as an important mediator of resistance to AR-targeted therapy and a driver of NEPC and lineage plasticity; their coordinated action and downstream signaling offers a potential novel therapeutic opportunity in late-stage PCa.

Endothelial Nucleoporin93 (Nup93) Maintains Vascular Function via Sun1-Dependent Regulation of RhoA-eNOS Signaling

Nguyen, T. D. — 2025-07-21

As the innermost lining of blood vessels, endothelial cells (ECs) regulate blood flow, maintain vascular tone, and limit inflammation for vessel health. EC-derived nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), is a vasodilator essential for improving blood flow and vascular homeostasis. The RhoA/ROCK pathway regulates eNOS levels, where overactivation decreases eNOS expression and downstream NO production. As such, RhoA/ROCK hyperactivity and increased pMLC have been identified as major contributors to age-associated vasoconstriction and hypertension. Intriguingly, recent studies identify Sun1, a key component of the linker of nucleoskeleton and cytoskeleton (LINC) complex, as a major regulator of RhoA/ROCK activity. Moreover, endothelial aging deteriorates nuclear pore complexes (NPCs) (i.e. nucleoporin [Nup93]) and impairs nucleocytoplasmic transport, thereby insinuating a role for nuclear envelope components in vessel homeostasis. Here, we show that targeted loss of endothelial Nup93 in adult mice significantly reduces eNOS expression and NO bioavailability for consequent defects in NO-dependent vasodilatory responses. In vitro knockdown of Nup93 in primary human ECs also decreases both eNOS expression and NO production. Mechanistically, we find that loss of Nup93 significantly reduces endothelial Sun1 levels for a concomitant increase in RhoA activity. Indeed, restoring Sun1 protein levels in Nup93-deficient ECs mitigates RhoA activity to rescue both eNOS expression and NO production. Taken together, we demonstrate endothelial Nup93, through Sun1 stabilization, as a novel regulator of eNOS-NO signaling and vessel reactivity, contributing to the growing importance of nuclear membrane components in EC and vascular biology.

DirectContacts2: A network of direct physical protein interactions derived from high-throughput mass spectrometry experiments

Claussen, E. R. — 2025-07-22

Cellular function is driven by the activity proteins in stable complexes. Protein complex assembly depends on the direct physical association of component proteins. Advances in macromolecular structure prediction with tools like AlphaFold and RoseTTAFold have greatly improved our ability to model these interactions in silico, but an all-by-all analysis of the human proteomes ~200M possible pairs remains computationally intractable. A comprehensive cellular map of direct protein interactions will therefore be an invaluable resource to direct screening efforts. Here, we present DirectContacts2, a machine learning model that distinguishes direct from indirect protein interactions using features derived from over 25,000 mass spectrometry experiments. Applied to ~26 million human protein pairs, our model outperforms previous resources in identifying direct physical interactions and enriches for accurate structural models including ~2,500 new AlphaFold3 models. Our framework enables structural modeling of disease-relevant complexes (e.g. orofacial digital syndrome (OFDS) complex) offering insights into the molecular consequences of pathogenic mutations (OFD1) and broadly, establishes a highly accurate protein wiring diagram of the cell.

Geographic distribution of genetic diversity of Heterocephalus glaber analyzed using whole genome sequencing and a chromosome-scale genome assembly

Wright, K. M. — 2025-07-26

Naked mole-rats (Heterocephalus glaber) are a species of rodent endemic to the Horn of Africa, notable among mammals for their long lifespans, resistances to a variety of stresses, and eusocial mating behavior. Though their natural range extends across large portions of Kenya, Ethiopia, Somalia, and Djibouti, the large majority of genetic and genomic analyses focus on Kenyan specimens. Here, we constructed a chromosome-scale reference genome assembly for H.glaber, along with new reference assemblies of both the Damaraland mole-rat (Fukomys damarensis) and guinea pig (Cavia porcellus) genomes to aid annotation. We leveraged our H.glaber assembly, along with modern whole-genome sequencing, to characterize the genetic diversity of specimens deriving from Kenya, southern Ethiopia, and eastern Ethiopia. We found the Kenyan and southern Ethiopian specimens to be closely related to each other and highly diverged from eastern Ethiopian specimens. We also found specimens collected from nearby locations in southern Ethiopia to be more closely related to Kenyan specimens than to each other. This unexpected distribution of shared genetic diversity highlights the importance of local migration barriers to gene flow in wild H.glaber populations.

Amyloid precursor protein mediates deficits in corticogenesis in Down syndrome cortical organoids

Rakowiecki, K. — 2025-07-27

Down syndrome (DS), due to trisomy 21 (T21), occurs in approximately 14.14 per 10,000 live births in the United States. Reduced neural progenitor cell (NPC) proliferation, delayed neurogenesis, impaired cortical lamination and altered cell fate specification are thought to contribute to cognitive impairments in DS individuals. The molecular mechanisms underlying these deficits are not fully understood. Notably, Amyloid precursor protein (APP), located on human chromosome 21 (HSA21), has been extensively implicated in these processes. Mouse models only partially recapitulate DS phenotypes due to genetic, developmental, and species-specific differences. Recent advances in induced pluripotent stem cell (iPSC) derived 3D cortical organoids allow for the study of DS cortical development in a human model system. Here, we show that normalizing APP gene copy number in DS cortical organoids ameliorated deficits in NPC proliferation, neuronal differentiation, and transcriptional programs. Our results demonstrate the value of cortical organoids in uncovering gene-specific roles in DS pathogenesis and identify APP as a promising target for addressing early neurodevelopmental impairments.

Comparative proteomic analysis of the ECM composition of the human omentum and mesentery, the main sites of ovarian cancer metastasis

Considine, J. M. — 2025-07-31

Due to its limited symptoms, high-grade serous ovarian cancer (HGSOC) has frequently metastasized extensively throughout the peritoneal cavity prior to its diagnosis, resulting in an overall five-year survival rate of less than 50%. The greater omentum and the small bowel mesentery are two of the most common metastatic sites in advanced HGSOC. However, the mechanisms underlying HGSOC metastatic tropism remain unknown. The extracellular matrix is a complex and dynamic meshwork of proteins that provides biochemical and mechanical signals to surrounding cells and has been shown to drive the dissemination of several cancer types to preferential distant sites. Here, using histological assessment and proteomics, we examined the composition of the extracellular matrix of paired omentum and mesentery samples from disease-free adult females. We found that the fibrillar collagen content of the mesothelial layer of the omentum was significantly higher than that of the mesentery. Using ECM-focused proteomics, we further defined the ECM composition - or matrisome - of these two tissues. We found that over 90% of the proteins detected were shared between the omentum and mesentery. Our analysis also revealed small subsets of tissue-specific ECM proteins. Future work will aim to test the possible functional contributions of these ECM proteins to HGSOC metastatic tropism. To facilitate the reuse of our dataset, we have deposited the raw mass spectrometry data and accompanying metadata files to the ProteomeXchange Consortium with the dataset identifier PXD061586.

Selectivity of the time-dependent M. tuberculosis LeuRS inhibitor ganfeborole is driven by target vulnerability

Wang, M. — 2025-08-03

Ganfeborole (GSK3036656) inhibits the Mycobacterium tuberculosis leucyl-tRNA-synthetase (mtLeuRS) and is in phase 2a clinical trials for the treatment of tuberculosis. Here we show that ganfeborole is a time-dependent inhibitor of mtLeuRS (IC50 1 nM) and generates a post-antibiotic effect of 77 h at 50xMIC (MIC 0.058 M) with M. tuberculosis H37Rv (Mtb), indicating that mtLeuRS is a highly vulnerable drug target and supporting the excellent in vivo efficacy of the drug. Ganfeborole is also a potent time-dependent inhibitor of Escherichia coli LeuRS (ecLeuRS, IC50 2 nM), however no antibacterial activity is observed towards E. coli up to 1 mM ganfeborole despite the observation that less potent ganfeborole analogs have antibacterial activity. To rationalize this observation, we propose that ganfeborole forms a complex with AMP that binds to the ecLeuRS editing site but does not impact aminoacylation. In support, addition of 12.5 M norvaline generates a ganfeborole MIC of 0.4 M since ecLeuRS is unable to hydrolyze norvaline-tRNALeu. Additionally, mutations that reduce the affinity and residence time of ganfeborole-AMP on ecLeuRS result in antibacterial activity. We propose that the activity of ganfeborole towards Mtb is because mtLeuRS is a highly vulnerable target so that only low levels of enzyme need to be inhibited by the ganfeborole-tRNALeu complex in contrast to ecLeuRS, which we previously demonstrated is a low vulnerability target.

Reversible therapeutic resistance in EGFR mutant lung cancer caused by RB1 loss induced lineage plasticity

Zhang, L. — 2025-08-06

Treatment with EGFR tyrosine kinase inhibitors (EGFR TKI) is standard of care for patients with lung cancer initiated by activating mutations in the EGFR gene. While EGFR TKI treatment is effective, virtually all patients progress on therapy. Loss of function mutations in the RB1 tumor suppressor gene are associated with poor EGFR TKI outcomes, but underlying mechanisms remain unclear. Analysis of genetically engineered experimental models and human clinical specimens described here demonstrates RB1 loss has minimal impact on initial cell cycle responses to EGFR TKI but markedly accelerates acquired resistance over time. RB1 loss promotes transcriptional plasticity that enables rare cells to spontaneously assume reversible, resistance-conferring lineage states. These findings highlight the importance of non-genetic adaptations in driving acquired EGFR TKI resistance, advance our understanding of how RB1 impacts cancer, and identify opportunities to improve EGFR TKI treatment outcomes by targeting reversible epigenetic resistance mechanisms.

Compact Vision--Language Models Enable Efficient and Interpretable Automated OCT Analysis Through Layer Specific Multimodal Learning

Haghighi, T. — 2025-08-11

Translating the intricate anatomical signatures of retinal disease from OCT B-scans into clear, accurate clinical narratives demands AI models that seamlessly fuse visual features with domain expertise. We curated a multimodal dataset of 40,000 OCT B-scans from public repositories and private clinical cohorts, each paired with expert validated summaries spanning six conditions: diabetic macular edema, diabetic retinopathy, geographic atrophy, drusen, choroidal neovascularization, and healthy retina. We introduce LO-VLM, a compact (247M parameter) vision-language model (VLM) that infuses anatomical guidance into both encoder and decoder for free form summary generation and multiclass disease classification. Benchmarking against state-of-the-art RetinaVLM, LLaVA-Med, and a ViT vision only model demonstrates superior performance. In a blinded evaluation by three board certified retina specialists scored the generated summaries, LO-VLM narratives achieved mean = 8.5 (standard deviation = 1.15) out of 10, compared to mean = 5.5 (standard deviation = 1.13) for RetinaVLM (p < 0.0001). In quantitative evaluations, LO-VLM achieved an SBERT similarity of 0.803 and a BERTScore F1 of 0.715, representing improvements of 8.2% and 28.8% over specialized VLM baselines. For disease classification, LO-VLM reached 96% accuracy (F1 = 96%), outperforming ViT by 13% and exceeding medical VLM benchmarks by over 62%. By reconciling interpretability with computational efficiency, LO-VLM establishes a new paradigm for efficient AI models in OCT interpretation.

Neuroinflammation in Olfactory Circuits Underlies Odor Hypersensitivity in a Rodent Model of Depression

Belonio, K. C. — 2025-08-19

Olfactory anhedonia and heightened aversion to unpleasant odors are well-documented features of depression in humans, yet the neural mechanisms linking chronic stress to altered olfactory perception remain poorly understood. We used the Unpredictable Chronic Mild Stress (UCMS) paradigm to examine how chronic stress affects olfactory avoidance behavior and glial cell morphology across multiple olfactory brain regions in male and female mice. UCMS-treated mice showed increased avoidance of aversive odorants in an odorized light/dark box assay, consistent with heightened aversive reactivity to odors following chronic stress. Using immunohistochemistry, we assessed microglial morphology and astrocyte density across six olfactory and limbic brain regions. Chronic stress produced region-specific glial remodeling: astrocyte counts were selectively elevated in the medial amygdala, and microglial process complexity was increased in the anterior olfactory nucleus and anterior piriform cortex. Microglial morphological complexity in the anterior piriform cortex was correlated with individual odor avoidance scores. These findings reveal that chronic stress induces regionally specific glial plasticity within olfactory sensory and affective networks and suggest that microglial remodeling in piriform cortex may contribute to stress-related changes in olfactory perception. Significance StatementChanges in sensory perception frequently accompany depression. While previous studies have implicated neuroinflammation in depression-related dysfunction within cortical and limbic structures, little is known about how chronic stress affects glial cells in olfactory processing regions. Here, we show that chronic stress induces glial remodeling in key olfactory areas, including the olfactory bulb, anterior piriform cortex, and medial amygdala, and that these changes correlate with heightened behavioral avoidance of aversive odors. These findings suggest that glial plasticity in sensory networks contributes to affective alterations in olfactory perception, revealing a potential mechanism by which mood disorders can influence sensory experience. This work advances our understanding of the neuroimmune basis of sensory-affective integration.

Persistent Immune Dysregulation during Post-Acute Sequelae of COVID-19 is Manifested in Antibodies Targeting Envelope and Nucleocapsid Proteins

Kwissa, M. — 2025-08-19

Post-Acute Sequelae of SARS-CoV-2 infection (PASC) syndrome or "Long COVID" represents a widespread health challenge that necessitates the development of novel diagnostic approaches and targeted therapies that can be readily deployed. Immune dysregulation has been reported as one of the hallmarks of PASC, but the extent of PASC immune dysregulation in patients over time remains unclear. We therefore assessed SARS-CoV-2-specific antibody responses, peripheral immune cell profiles, autoantibody profiles and circulating cytokines for up to 6 months in participants with a SARS-CoV-2 infection who either convalesced or developed PASC. Compared to convalescent, PASC participants with a broad range of PASC phenotypes exhibited persistently elevated IgG titers for SARS-CoV-2 Envelope and Nucleocapsid proteins over the 6 months of study duration. In contrast, the IgG responses to Spike protein were significantly lower in the PASC cohort with predominantly IgG1 and IgG3 class-switched bias. Using CyTOF analysis, we show elevated numbers of circulating T follicular helper cells (cTFH) and mucosa-associated invariant T cells (MAIT), which also correlated with high anti-Envelope IgG titers. Persistent immune activation was accompanied by augmented serum cytokine profiles with LIF, IL-11, Eotaxin-3, and HMGB-1 in PASC participants, who also demonstrated significantly higher rates of autoantibodies. These findings highlight the persistence of immune dysregulation in PASC, underscoring the need to explore targeted therapies addressing viral persistence, dysregulated antibody production, and autoimmunity.

Missing data in single-cell transcriptomes reveals transcriptional shifts

Chen, R. — 2025-08-21

Profiling thousands of single cell transcriptomes is routine, yet cell prioritization based on response to biological perturbations is challenging and confounded by clustering, normalization and dimensionality reduction strategies. We developed a scoring approach independent of these obstacles that unbiasedly identifies distinct transcriptomes within a set based on missing data patterns, allowing cell prioritization and feature selection for downstream analysis. Our method applied to D. discoideum reveals a metabolic shift that marks the transition between the amoeboid and aggregated states of this model organism.

Ceramide-induced Endoplasmic Reticulum Stress as a Targetable Vulnerability in Endocrine Therapy-Resistant Breast Cancer

Pal, P. — 2025-08-22

Despite the success of endocrine therapy (ET) in treating hormone receptor-positive breast cancer, a significant proportion of patients relapse during or after treatment, making ET resistance a major clinical challenge. Previously we have shown that ET-resistant breast cancer cells exhibit reduced ceramide levels and an increased sensitivity to ceramide-induced cell death. Here, we demonstrate that ceramides induce a distinct transcriptional reprogramming in ET-resistant cells, characterized by upregulation of endoplasmic reticulum stress (EnRS) pathways. Ceramide-induced EnRS is PERK-dependent and functionally linked to cell death in multiple models of ET resistance. Using a photoactivatable ceramide probe, we identify TRAM1 as a functionally important ceramide-interacting protein (CIP) in ET-resistant cells that correlates with worse relapse-free survival and a more aggressive breast cancer phenotype in luminal breast cancer patients. Additionally, knockdown of TRAM1 phenocopies ceramide action in ET resistance, thereby suggesting its role in mediating ceramide-induced lethal actions in ET resistance. Together, our findings reveal that ET-resistant breast cancer cells are more sensitive to PERK-mediated EnRS as compared to their ET-sensitive counterparts. Ceramides can exploit this dependence by interacting with CIPs such as TRAM1, leading to PERK activation and consequential cell death preferentially in the ET-resistant breast cancer models.

In-depth proteomic profiling of the extracellular matrix of pancreatic ductal adenocarcinomas identifies signatures correlating with lymphocyte infiltration

Considine, J. M. — 2025-08-27

The extracellular matrix (ECM) is a complex assembly of proteins surrounding cells. It is a critical component of the tumor microenvironment that plays an active role in tumor progression and modulation of tumor response to treatment. Pancreatic ductal adenocarcinoma (PDAC) is a cancer type characterized by one of the worst prognoses, as it is often diagnosed at an advanced stage. It is also characterized by a very dense ECM, which hinders efficient drug delivery. In addition, PDACs are considered "cold" tumors as they fail to elicit a strong immune response, challenging the use of immunotherapy for PDAC cancer patients. Yet, the interplay between the ECM and immune cells within the PDAC tumor microenvironment remains poorly understood. Here, we employed ECM-focused proteomics to profile the ECM compositions of PDAC mouse models characterized by different levels of CD8+ T-cell infiltration. We found that CD8lo, or "cold" tumors, and CD8hi, or "hot" tumors, exhibited different ECM compositions. Interrogation of publicly available single-cell RNA-sequencing datasets of human PDACs further revealed that the ECM proteins distinguishing hot and cold PDACs are secreted by multiple stromal cell populations, including cancer-associated fibroblasts, stellate cells, and macrophages. Last, we found that the expression of a subset of the genes encoding ECM proteins characteristic of the CD8lo phenotype correlated with CD8+ T-cell infiltration in human PDAC samples and patient survival. This study paves the way for the development of ECM-modulating interventions to enhance immune cell infiltration and responsiveness to immunotherapy. SignificanceWe report the identification of ECM protein signatures correlating with the level of CD8+ lymphocyte infiltration in murine models of pancreatic ductal adenocarcinomas and human samples. This work paves the way for the development of ECM-modulating therapeutic strategies to enhance lymphocyte infiltration and, hence, the efficacy of immunotherapies.

Multiomic Analysis Reveals Molecular Pathways Associated with Intestinal Aggregation of α-Synuclein

Balsamo, J. M. — 2025-08-30

Aggregates of the protein -synuclein may initially form in the gut before propagating to the brain in Parkinsons disease. Indeed, our prior work supports that enteroendocrine cells, specialized intestinal epithelial cells, could play a key role in the development of this disease. Enteroendocrine cells natively express -synuclein and synapse with enteric neurons as well as the vagus nerve. Severing the vagus nerve reduces the load of -synuclein aggregates in the brain, suggesting that this nerve is a conduit for gut-to-brain spread. Enteroendocrine cells line the gut lumen, as such, they are in constant contact with metabolites of the gut microbiota. We previously found that when enteroendocrine cells are exposed to nitrite--a potent oxidant produced by gut bacterial Enterobacteriaceae--a biochemical pathway is initiated that results in -synuclein aggregation. Here, we determined that dopamine production is critical to this mechanism of nitrite-induced -synuclein aggregation. Using enteroendocrine cells, we modulated dopamine biosynthesis and profiled the cellular proteome and lipidome. Proteomic signatures in dopamine-free cells were distinctly different than in enteroendocrine cells, highlighting pathways relevant to intestinal development of Parkinsons disease. Intriguingly, we observed that enteroendocrine cells maintain viability upon exposure to nitrite and in the presence of -synuclein aggregates. This cellular robustness suggests that dopamine-producing enteroendocrine cells may be a reservoir of toxic -synuclein aggregates, which can spread through a prion-like process. As a possible antidote, our findings show that benserazide--a chemical inhibitor of dopamine biosynthesis--limits formation of these aggregates in enteroendocrine cells. These studies lay a foundation for mechanistically informed therapeutic targets to prevent intestinal formation of -synuclein aggregates before they spread to the brain. For Table of Contents Use Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/672706v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1668b83org.highwire.dtl.DTLVardef@1515382org.highwire.dtl.DTLVardef@41b1d2org.highwire.dtl.DTLVardef@1018049_HPS_FORMAT_FIGEXP M_FIG C_FIG

UALCAN Mobile, an app for cancer proteogenomic data analysis.

Varambally, S. — 2025-09-05

Cancer is a complex disease affecting various organs and is a major cause of death worldwide. During cancer initiation, disease progression, and tumor metastasis, various genomic and proteomic alterations are observed. Recent technological advances have led to the generation of large amounts of molecular data, including genomics and transcriptomics. These large-scale datasets can be utilized to analyze and identify sub-class-specific cancer biomarkers and targets. However, there is a need for the development of user-friendly tools for large-scale data analysis, disseminating the analyzed data in a visualizable format to cancer researchers with no programming skills. We developed UALCAN, a comprehensive platform that allows users to integrate disparate data to better understand the genes, proteins, and pathways perturbed in cancer and make discoveries of potential biomarkers and targets. In the current study, we describe the development of the UALCAN Mobile application (app) that will provide cancer transcriptomic data obtained from The Cancer Genome Atlas (TCGA) project to evaluate protein-coding gene expression based on various stratifications, including stage, grade, race, gender, and molecular-subtypes across over 30 types of cancers. In addition, the UALCAN mobile provides data analysis options for epigenetic changes due to DNA promoter methylation and Clinical Proteomic Tumor Analysis Consortium (CPTAC) cancer proteomic data. The app provides access to large cancer molecular datasets on the go. To find changes in the expression of causative genes and proteins and to identify biomarkers and therapeutic targets, UALCAN mobile app will be extremely valuable. The "UALCAN Mobile" app is free to use and can be downloaded from both the iOS/Apple and the Android Play Store and has been downloaded over 100 times in each of iOS and android app stores. Key PointsO_LICancer, a complex disease affecting various organs, is a major cause of death worldwide. C_LIO_LIMany molecular alterations, gene and protein expression changes lead to the initiation of cancer, disease progression, and tumor metastasis. C_LIO_LIThe available large genomic and proteomics datasets can be utilized to analyze and identify sub-class-specific cancer biomarkers and targets with innovative tools. C_LIO_LIUALCAN Mobile application (app) will provide gene expression, promoter DNA methylation, and protein expression across cancers based on various stratifications, including stage, grade, and molecular subtypes across over 30 types of cancers. C_LIO_LIUALCAN Mobile app is free and can be downloaded from both iOS/Apple and the Android Play Store. C_LI

MiR-34a deficiency enhances nucleic acid sensing and type I IFN signaling in a mouse model of Alzheimer's disease

Yang, J. — 2025-09-09

MiR-34a is implicated in aging, cell senescence, inflammation, and neurodegenerative diseases. In order to investigate the role of miR-34a in Alzheimers disease (AD), we produced an AD mouse model, Tg-SwDI mice, with whole body/constitutive miR-34a knockout (KO). MiR-34a KO improved long-term memory in Tg-SwDI mice, which was associated with decreases in the ratio of insoluble A{beta}42 to A{beta}40 and with increases in soluble and insoluble A{beta}40 in the cerebral cortex. Anti-Iba1 immunofluorescence revealed increases in activated microglia. Bulk RNA-sequencing of the hippocampus followed by a gene set enrichment analysis (Enrichr) identified "cellular response to type I interferon" and "type I interferon signaling pathway" as the most prominent gene sets in miR-34a KO Tg-SwDI mice compared to miR-34a wild-type Tg-SwDI mice. Many interferon-stimulated genes (ISGs) that characterize interferon responsive microglia (IRM) were upregulated in miR-34a KO Tg-SwDI mice. MiR-34a knockdown strongly enhanced ISGs expression in TLR7 ligand-stimulated BV2 microglia. These results suggest that miR-34a inhibits the transition of microglia to the IRM state that may modulate synaptic and cognitive functions in neurodegenerative diseases and aging.

A regulator of amino acid catabolism controls Acinetobacter baumannii gut colonization

Geary, J. H. — 2025-09-11

Asymptomatic gut colonization increases the risk of clinical infection and transmission by the multidrug-resistant pathogen Acinetobacter baumannii. Ornithine utilization was shown to be critical for A. baumannii competition with the resident microbiota to persist in gut colonization, but the regulatory mechanisms and cues are unknown. Here, we identify a transcriptional regulator, AstR, that specifically activates the expression of the A. baumannii ornithine utilization operon astNOP. Phylogenetic analysis suggests that AstR was co-opted from the Acinetobacter arginine utilization ast(G)CADBE locus and is specialized to regulate ornithine utilization in A. baumannii. Reporter assays showed that astN promoter expression was activated by ornithine but inhibited by glutamate and other preferred amino acids. astN promoter expression was similarly activated by incubation with fecal samples from conventional mice but not germ-free mice, suggesting AstR-dependent activation of the astN promoter responds to intermicrobial competition for amino acids. Finally, AstR was required for A. baumannii to colonize the gut in a mouse model. Together, these results suggest that pathogenic Acinetobacter species evolved AstR to regulate ornithine catabolism, which is required to compete with the microbiota during gut colonization.

Protection against diet-induced obesity by a single-point mutation in Kir2.1 channels

Beverley, K. M. — 2025-09-17

High-fat diet (HFD)-induced obesity remains a significant global health challenge. In this study, we show that a global knock-in CRISPR mouse with the Kir2.1L222I single-point mutation exhibits remarkable resistance to HFD-induced obesity. We identify palmitic acid (PA), a prevalent long-chain fatty acid in obesity, as a novel negative regulator of Kir2.1. Kir2.1L222I previously shown to protect against cholesterol-mediated inhibition of Kir2.1, also confers protection against PA-induced suppression. Moreover, PA-induced suppression of Kir2.1 results in a significant loss of flow-induced vasodilation (FIV), while the L222I mutation exerts a protective effect. Notably, Kir2.1L222I mice display significant protection against HFD-induced weight gain and adiposity independent of caloric intake. Specifically, the mutant mice show increased lean mass and decreased fat mass, specifically in both visceral and subcutaneous white adipose tissue (WAT) and intrascapular brown adipose tissue (BAT). Importantly, visceral-to-subcutaneous white adipose ratios decrease while BAT/WAT tissue ratios increase, suggesting a metabolically favorable fat distribution. This protection correlates with enhanced physical activity and increased energy expenditure. Metabolomic analysis reveals elevated TCA cycle metabolites in adipose tissue of Kir2.1L222I mice, consistent with their enhanced energy expenditure. These findings highlight Kir2.1 channels as potential therapeutic targets for obesity and related metabolic disorders.

Fast Ripple-Delta Coupling as Early Biomarker for Post-Traumatic Epileptogenesis in Repetitive Brain Injury

Shandra, O. — 2025-09-17

Traumatic brain injury (TBI) can induce post-traumatic epilepsy (PTE), but early biomarkers for epileptogenesis are lacking. We used a repetitive diffuse TBI (rdTBI) model in mice with continuous video-EEG monitoring up to 4[1/2] months post-injury to investigate electrographic biomarkers before and during post-traumatic seizure development. 25% of mice developed post-traumatic seizures with highly variable latency (5-126 days post-injury). Most significantly, we identified fast ripple-delta DOWN state coupling as an early biomarker that was detectable at 4 days post-TBI and appeared before seizure onset in all seizure-experiencing mice. This EEG signature distinguished seizure-experiencing from seizure-free TBI mice with high specificity. Power spectrum analysis revealed elevated delta and theta power, reduced physiological fast oscillations (alpha, beta, gamma) and increased pathological high-frequency oscillations (fast ripples) in seizure-experiencing animals, indicating network hyperexcitability. Spike analysis showed that while TBI itself increased cortical excitability, seizure onset triggered a dramatic further escalation in interictal activity. These electrographic signatures were remarkably consistent across all seizure-experiencing animals regardless of single or recurrent seizure pattern. Our results demonstrate that fast ripple-delta coupling represents a promising early biomarker detectable at 4 days post-TBI, before seizure onset, offering potential for early identification of post-traumatic seizure susceptibility. Importantly, this biomarker identified all seizure-prone animals regardless of whether they developed single or recurrent seizures, suggesting shared underlying mechanisms and clinical relevance for any post-traumatic seizure occurrence. These findings emphasize the utility of temporal EEG analysis for detecting early electrographic changes in post-traumatic epileptogenesis and may inform future intervention strategies. Key PointsO_LIFast ripple-delta DOWN state coupling was detectable as early as 4 days post-TBI and appeared before seizure onset in seizure-experiencing mice, representing the first early biomarker that can stratify animals for epileptogenesis risk during the critical latent period. C_LIO_LIDelta and theta power increased while alpha, beta and gamma power decreased in all seizure-experiencing mice post-TBI, creating a consistent electrographic signature regardless of whether animals developed single or recurrent seizures. C_LIO_LIFast ripples were elevated and gamma-to-HFO ratios were reduced in seizure-experiencing mice, reflecting network hyperexcitability shift and potential inhibitory dysfunction that preceded seizure onset. C_LIO_LISeizure onset triggered a 3-fold escalation in spike activity, while baseline spike differences between TBI and pre-seizure mice were not significant, highlighting the limitation of spike counts alone as predictive biomarkers during the latent period. C_LIO_LIElectrographic signatures were almost similar across all seizure patterns (single and recurrent), suggesting shared underlying mechanisms of network dysfunction, though larger studies are needed to determine if biomarkers can predict seizure frequency in addition to seizure susceptibility. C_LI

LRH-1 is a novel regulator of neutrophil-driven immune responses within the tumor microenvironment.

Wang, Y. — 2025-09-18

Elevated plasma cholesterol levels have been linked to worse outcomes in breast and ovarian cancer. Prior work including our own has demonstrated that myeloid immune cells are highly responsive to cholesterol fluctuations and to proteins involved in cholesterol regulation. However, the specific roles of Liver Receptor Homolog-1 (LRH-1, or NR5A2), a key transcriptional regulator of cholesterol homeostasis, within myeloid cells remains largely undefined. Interestingly, LRH-1 mRNA levels are reduced in both breast and ovarian tumors compared to normal tissue. Its elevated expression within tumors is associated with increased survival time. These clinical correlations prompted us to explore the role of LRH-1 in myeloid cells particularly in the context of breast and ovarian cancer progression. Initial analyses confirmed LRH-1 expression in various myeloid cell types, with particularly high levels in neutrophils. We therefore focused on how LRH-1 influences neutrophil behaviors relevant to cancer, including migration, NETosis, phagocytosis, and interactions with T cells. Small molecule ligands for LRH-1 regulated neutrophil migration towards cancer cells. Phagocytosis was also regulated by LRH-1 small molecule ligands, the extent being dependent on type of bait (e. coli vs. cancer cells). In T cell co-cultures, neutrophils pretreated with an LRH-1 agonist promoted greater T cell expansion - particularly in CD4 cells - while LRH-1 inhibition suppressed this response. Moreover, LRH-1 activation reduced NETosis, while treatment with an antagonist or inverse agonist enhanced NETosis. Treatment of mice with an inverse agonist of LRH-1 increased the growth of 4T1 and E0771 mammary tumors. Given prior evidence that neutrophils contribute to the recurrence of dormant lesions, we further examined the role of LRH-1 in this context. Mice harboring dormant D2.0R mammary cancer lesions treated with an LRH-1 inverse agonist exhibited earlier tumor recurrence and enhanced metastatic progression compared to vehicle-treated controls. In contrast, treatment with BL001, a small chemical LRH-1 agonist, delayed recurrence in D2.0R-grafted mice. Collectively, these findings reveal that LRH-1 is a novel regulator of neutrophil-driven immune responses within the tumor microenvironment. LRH-1 thus remerges as a promising therapeutic target to suppress metastasis or prevent recurrence in breast cancer.

Broad-Spectrum HIV-1 Detection and Neutralization via Multivalent Designer DNA Nanostructures

Umrao, S. — 2025-09-21

Early and accurate detection of HIV-1 remains a critical unmet need, particularly during the acute phase of infection when viral loads are low and transmission risk is highest. Here, we report a modular diagnostic and antiviral platform based on designer DNA nanostructures engineered for high-affinity recognition of the HIV-1 envelope glycoprotein (GP120). A custom DNA aptamer, termed HINT, was developed to bind GP120 across major HIV-1 subtypes (Groups M and P; subtypes A and B) with nanomolar affinity. To amplify binding strength, HINT aptamers were spatially patterned onto a net-shaped DNA nanostructure (DNA-NetHINT) that geometrically matches the trimeric GP120 spikes on the viral surface. Using multivalent interactions, the nanostructure enabled up to 104-fold improvement in binding affinity (sub-picomolar KD), confirmed by surface plasmon resonance. Integration of DNA-NetHINT into a paper-based lateral flow assay produced a low-cost, saliva-compatible self-testing device capable of detecting intact HIV-1 virions at concentrations as low as 328 viral copies per test, outperforming commercial fourth-generation rapid diagnostic tests. In addition to its diagnostic capabilities, the DNA-NetHINT construct exhibited potent antiviral activity, reducing pseudovirus infection with an EC50 of [~]1.8 nM, nearly 1,000-fold more effective than free aptamers. This work demonstrates a dual-function DNA nanotechnology platform that enables both ultrasensitive HIV-1 detection and entry inhibition. The approach is broadly applicable to other enveloped viruses and represents a promising step toward next-generation molecular theranostics for infectious disease management.

Compositional and Functional Profiling of the Gut Microbiome in Sarcoidosis

Lee, J. M. — 2025-09-24

BackgroundSarcoidosis is a chronic inflammatory disease characterized by systematic granuloma formation, predominantly in the lungs. One leading hypothesis posits that prolonged exposure to microbial antigens may trigger chronic and dysregulated inflammation. Prior studies have examined the lung microbiota of sarcoidosis patients, but the role of the gut microbiota along the gut-immune axis remains largely unexplored. We elucidate the community composition and function of the gut microbiome in sarcoidosis. MethodsSubjects diagnosed with sarcoidosis (n=37) were recruited and matched with healthy control subjects (n=37). Stool samples were collected from sarcoidosis patients for metagenomic sequencing, followed by taxonomic classification and functional annotation as KEGG Orthology (KO) pathways. Differential analysis between sarcoidosis and control cohorts was performed using both standard and compositional approaches. ResultsSarcoidosis microbiomes exhibited similar alpha diversity but significantly different beta diversity from control microbiomes (p<0.05). At the phylum level, the proportion of Actinobacteria and Firmicutes expanded whereas Bacteroides and Verrucomicrobia proportionally shrunk in sarcoidosis gut microbiomes compared to healthy control ones. At the species level, 1,495 species were significantly more abundant and 279 less abundant in sarcoidosis compared to controls (FDR<0.05). Of these, 13 species demonstrated greater intergroup than intragroup variation (effect size>1), including microbes previously detected in the lungs and granulomas of sarcoidosis. Functional profiling revealed 409 KO pathways that were significantly underrepresented in sarcoidosis compared to control (FDR<0.05), of which 79 met an effect size > 1. These include pathways in amino acid and energy metabolism as well as HIF-1 signaling. ConclusionsOur findings highlight the potential role of gut microbiome composition and function in inflammatory processes that are associated with granuloma formation in sarcoidosis and warrant further investigation into gut hyperoxia-driven dysbiosis.

ChromPolymerDB: A High-Resolution Database of Single-Cell 3D Chromatin Structures for Functional Genomics

Chen, M. — 2025-09-29

The three-dimensional (3D) organization of chromatin plays a critical role in regulating gene expression and genomic processes like DNA replication, repair, and genome stability. Although these processes occur at the individual-cell level, most chromatin structure data are derived from population-averaged assays, such as Hi-C, obscuring the heterogeneity of single-cell conformations. To address this limitation, we developed a polymer physics-based modelling framework, the Sequential Bayesian Inference Framework (sBIF), that deconvolutes bulk Hi-C data to reconstruct single-cell 3D chromatin conformations. To support a broader use of sBIF, we created ChromPolymerDB, a publicly accessible, high-resolution database of single-cell chromatin structures inferred by sBIF. The database contains [~]108 reconstructed 5 kb-resolution single cell structures, spanning over 60,000 genomic loci across 50 human cell types and experimental conditions. ChromPolymerDB features an interactive web interface with tools for 3D structural analysis and multi-omics integration. Users can explore associations between chromatin conformation and gene expression, epigenetic modifications, and regulatory elements. The platform also supports comparative analyses to identify structural changes across cell types, developmental stages, or disease contexts. ChromPolymerDB offers a unique resource for researchers studying the relationship between genome architecture and gene regulation, and for advancing comparative 3D genomics. ChromPolymerDB is available online at https://chrompolymerdb.bme.uic.edu/. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/678816v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@110c807org.highwire.dtl.DTLVardef@bc713forg.highwire.dtl.DTLVardef@1af0ea9org.highwire.dtl.DTLVardef@1eba638_HPS_FORMAT_FIGEXP M_FIG C_FIG

A deep learning and co-conservation framework enable discovery of non-canonical Cas proteins

He, B. — 2025-10-01

CRISPR-Cas systems are central to prokaryotic adaptive immunity, widely harnessed for biotechnology. Yet, their vast and uncharacterized diversity, especially non-canonical variants, impedes full exploitation. Here we present BioPrinCRISPR, a class-agnostic computational framework leveraging gene co-conservation, protein domain co-occurrence, and embedding similarity to identify and characterize CRISPR-Cas systems across prokaryotic genomes. Applying BioPrinCRISPR to over one million bacterial genomes, we uncovered extensive canonical and uncharacterized systems, revealing a rich landscape of atypical Cas proteins and novel domain architectures. Notably, we identified recurrent fusion proteins with unique enzymatic combinations, suggesting roles in regulatory control or nucleic acid remodeling. Experimental validation of two divergent Cas13an-like effectors demonstrated RNA knockdown capacity in human cells, confirming our frameworks predictive power. These findings expand the functional repertoire of CRISPR-associated proteins and highlight unexplored modes of microbial immunity. BioPrinCRISPR thus stands as a powerful tool for comprehensively mapping CRISPR-Cas diversity, offering new insights into prokaryotic defense and facilitating discovery of novel candidates for next-generation genome engineering. An accompanying interactive web platform was also developed to facilitate data exploration.

Evolutionarily Conserved and Divergent Mechanisms of Dual Ca2+ Sensors in Synaptic Vesicle Exocytosis

Li, L. — 2025-10-01

Neurotransmitter release at the C. elegans neuromuscular junction is governed by a dual Ca{superscript 2} sensor system composed of SNT-1 and SNT-3, which are functional analogs of synaptotagmin-1 and -7 (Syt1/Syt7) in mammalian central synapses. In this study, we investigated how SNT-1 and SNT-3 mediate fast and slow neurotransmitter release through their potential interactions with the SNARE complex and their polybasic motifs. AlphaFold 3 models of SNT-1-SNARE and SNT-3-SNARE complexes accurately recapitulated the canonical Syt1 C2B-SNARE primary interface (Zhou et al., 2015, Nature) and precisely identified conserved binding residues within the C2B domains, as well as in SNAP-25 and Syntaxin, highlighting the evolutionary conservation of this interaction. Electrophysiological analyses using targeted mutagenesis demonstrated that both SNT-1 and SNT-3 require C2B-SNARE interactions and polybasic motifs within their C2 domains to drive evoked fast and slow neurotransmitter release. Notably, SNT-1 and SNT-3 exhibited differential dependence on distinct regions of the C2B-SNARE interface and their respective polybasic motifs, suggesting that Ca{superscript 2}-triggered fast and slow release operate via distinct mechanistic strategies. Furthermore, we found that SNT-1 mediates spontaneous neurotransmitter release through multiple pathways, involving not only the primary C2B-SNARE interface but also additional putative SNARE-binding interactions. Together, our findings uncover both conserved and divergent mechanisms for synaptic exocytosis regulated by the dual Ca2+ sensors in C. elegans.

Integrative analysis of spatiotemporal transcriptomics delineates dynamic cell states in squamous tumorigenesis

Sreekanth, V. — 2025-10-02

Squamous cell cancers are responsible for 1 in 5 cancer deaths and survival improvements lag behind those seen in adenocarcinomas. This disparity is in large part due to the limited impact of immunotherapy due to therapeutic resistance, where less than ten percent of patients respond in early stage squamous cell carcinomas of the Head and Neck. Mechanisms that govern intrinsic resistance remain poorly understood and likely arise during the premalignant or dysplastic state. Here, we generated a dataset of murine and human oral squamous epithelia spanning the earliest premalignant stages through invasive carcinoma. Integrative analysis of single-cell and spatial transcriptomics data across the dysplasia to carcinoma continuum reveals early and sustained shifts in epithelial transcriptomes. Spatially informed cell-cell interaction analysis reveals dysplasia-specific upregulation of wound healing and immune remodeling programs in severe dysplasia and invasive carcinoma in a human sample of oral dysplasia. The presence of these transcriptomic programs within early dysplasia may account for the aggressive clinical presentations of squamous cancers including intrinsic therapeutic resistance.

Towards Human Inverse Dynamics from Real Images: A Dataset and Benchmark for Joint Torque Estimation

Chen, C. — 2025-10-04

AO_SCPLOWBSTRACTC_SCPLOWHuman inverse dynamics is an important technique for analyzing human motion. Previous studies have typically estimated joint torques from joint pose images, marker coordinates, or EMG signals, which severely limit their applicability in real-world scenarios. In this work, we aim to directly predict joint torques during human movements from real human images. To address this gap, we present the vision-based inverse dynamics dataset (VID), the first dataset tailored for the joint torque prediction from real human images. VID comprises 63,369 frames of synchronized monocular images, kinematic data, and dynamic data of real human subjects. All data are carefully synchronized, refined, and manually validated to ensure high quality. In addition, we introduce a comprehensive benchmark for the vision-based inverse dynamics of real human images, consisting of a new baseline method and a new evaluation criteria with three levels of difficulty: (i) overall joint torque estimation, (ii) joint-specific analysis, and (iii) action-specific prediction. We further compare the baseline result of our VID-Network with other representative approaches, our baseline method achieves the state-of-the-art performance on almost all the evaluation criteria. By releasing VID and the accompanying evaluation protocol, we aim to establish a foundation for advancing biomechanics from real human images and to facilitate the exploration of new approaches for human inverse dynamics in unconstrained environments. The project website is https://github.com/MedVisionKitchen/VID-Benchmark.

Commensal Escherichia coli colonization triggers Peyer's patch development

Gerner, R. R. — 2025-10-07

The gut microbiota plays a pivotal role in shaping mucosal immunity, yet the specific microbes contributing to lymphoid tissue development remain poorly defined. Here, we identify Escherichia coli, a pioneer commensal bacterium, as a key driver of naive B cell accumulation in gut Peyers patches and lamina propria via a CXCR2-dependent mechanism. We show that E. coli promotes B cell recruitment through the production of curli amyloid fibers, which signal via Toll-like receptor 2 (TLR2). Notably, this effect extends beyond the neonatal period, revealing a broader temporal window for microbial modulation of mucosal immune development. These findings reveal a previously unrecognized role for a defined gut commensal bacterium and its molecular products in orchestrating the formation of gut-associated lymphoid tissue and B cell recruitment.

Biophysical simulations of fMRI responses using realistic microvascular models: insights into distinct hemodynamics in humans and mice

Hartung, G. — 2025-10-08

1Functional Magnetic Resonance Imaging (fMRI) is broadly used to measure human brain activity, however the hemodynamic changes that comprise the fMRI response to neuronal activity are often interpreted using microscopy data in mice. These microscopy data provide ground-truth observations of how individual blood vessels respond to neuronal activity and thus form the basis of our fundamental understanding of neurovascular coupling. Although these invasive experiments provide invaluable insight, there are striking differences in the vascular architecture of mouse and human brains that may influence the hemodynamic response. Motivated by this, we developed a biophysical modeling framework for realistic hemodynamic simulations in both mouse and human cerebral cortex. For this, we utilized Vascular Anatomical Network (VAN) models that explicitly represent the full microvascular tree as a single connected network, originally based on anatomical reconstructions from a given location of mouse cerebral cortex. We extended the VAN modeling framework using synthetic VAN models representing the microvascular network at a single location of the human cerebral cortex. To account for larger size and complexity of the human VAN models, we developed an efficient computational framework to simulate the full hemodynamic responses in this human model and compared the simulated fMRI responses between mice and humans. Our biophysical simulations are based entirely on first principles (e.g., conservation of mass); model parameter values were fixed across all simulations, not tuned to fit data, as they represent meaningful physical constants taken from previous measurements. Only two simple calibrations were tuned for each simulation, to match baseline perfusion rates (blood flow) and oxygen extraction (OEF). Our results show that differences in microvasculature indeed influenced the hemodynamic response and led to observable differences in timing--e.g., the simulated fMRI response peak in humans was delayed by [~]2 s compared to mice, consistent with prior fMRI observations. While there are many known differences in vascular architecture in rodents and humans, we also discovered that, unexpectedly, an asymmetry in the numbers of branches of the penetrating intracortical arterioles and venules appears to be conserved across species. We demonstrate through further simulations that this anatomical property may also be needed for suitable hemodynamic responses. Our framework thus provides a valuable tool for bridging in-vivo microscopy of microvascular dynamics to human fMRI.

Mechanical stretch disrupts calcium dynamics and redistributes Piezo1 in human astrocytes

Shiravi, S. — 2025-10-09

Astrocytes regulate the activity of nearby neurons so disruption of astrocyte calcium dynamics by traumatic brain injury (TBI) could have profound consequences for neural network activity in the brain. In this study, human induced pluripotent stem cell (hiPSC)-derived astrocytes were used in a two-dimensional (2D) in vitro stretch injury model to evaluate the effect of trauma on calcium dynamics, mitochondrial function, and the mechanosensitive ion channel Piezo1. Outcomes were assessed using live imaging, immunostaining, and RNA sequencing. Cell viability, mitochondrial membrane potential, and spontaneous calcium transients declined as injury severity increased. At moderate injury severity, the decreases in mitochondrial membrane potential and calcium dynamics were temporary. The spatial distribution of Piezo1 also changed temporarily after injury. RNA sequencing identified 196 genes that changed expression after injury, including downregulation of mitochondrial and oxidative metabolic processes and upregulation of cortical thinning pathways. These findings establish this model as a platform for investigating the cellular mechanisms of TBI and its influence on neurodegeneration.

CSCN: Inference of Cell-Specific Causal Networks Using Single-Cell RNA-Seq Data

Wang, M. — 2025-10-09

Understanding gene regulation is fundamental to deciphering the coordinated activity of genes within cells. Although single-cell RNA sequencing (scRNA-seq) enables gene expression profiling at cellular resolution, most gene network inference methods operate at the tissue or population level, thereby overlooking regulatory heterogeneity across individual cells. Recent approaches, such as Cell-Specific Network (CSN) and its extension c-CSN, attempt to construct gene networks at single-cell resolution, providing a more detailed view of the regulatory logic underlying individual cellular states. However, these methods remain limited by high false positive rates due to indirect associations and lack of directionality or causal interpretability. To address these issues, we propose the Cell-Specific Causal Network (CSCN) framework, which infers directed, cell-specific gene regulatory relationships by explicitly modeling causality. CSCN combines causal discovery techniques with efficient computation using kd-trees and bitmap indexing to perform conditional independence testing, yielding sparse and interpretable causal graphs for each cell that effectively suppress indirect and spurious associations. We demonstrate through simulations that CSCN significantly reduces false positives compared to existing methods. Furthermore, we evaluate the quality of the inferred causal networks via clustering on the Causal Katz Matrix (CKM), and CSCN outperforms CSN and c-CSN in distinguishing cellular states.

An extremophilic Nocardiopsis strain from Great Salt Lake expands the taxonomic range of mycolic acid biosynthesis

Scott, A. F. — 2025-10-12

Mycolic acids, long-chain fatty acids that form the characteristic and relatively impermeable mycomembrane, have long been considered a defining chemotaxonomic feature of the order Mycobacteriales. Here, we report that Nocardiopsis bonnevillensis, a new type strain isolated from the hypersaline environment of Great Salt Lake, is the first organism outside of this order known to produce mycolic acids. Lipid profiling, acid-fast staining, isoniazid sensitivity, and genome mining confirmed hallmark features of mycolic acid biosynthesis. Evaluation of the strains metabolic capabilities led to the isolation of bonnevanoside, a thiophenyl nonulopyranoside reported here for the first time from a natural source. Moreover, additional Great Salt Lake-derived Nocardiopsis isolates exhibited acid-fast staining, suggesting that this trait may be more widespread than previously recognized. Altogether, these findings expand the taxonomic distribution of mycolic acid biosynthesis, challenge long-standing chemotaxonomic boundaries, and highlight the potential ecological significance of mycolate-containing envelopes in supporting bacterial survival in extreme environments.

Patterns of Brain Activation and Hippocampal Functional Connectivity Supporting Verbal Memory in Midlife Women

Wugalter, K. A. — 2025-10-14

Women show declines in verbal memory across the menopause transition that may persist into the postmenopause. The goal of the present study was to characterize the patterns of brain activity and hippocampal functional connectivity that support verbal memory performance in midlife postmenopausal women. The study sample included 171 midlife postmenopausal women from the MsBrain I study (mean age = 59.3 years, mean education= 15.7 years, 87.7% white). All participants were cognitively normal, native English speakers, not taking menopausal hormone therapy. Participants completed neuropsychological (California Verbal Learning Test [CVLT]) and neuroimaging assessments, including an fMRI task of verbal encoding and recognition. Findings indicated that during verbal encoding, greater activation of bilateral prefrontal and medial temporal regions, as well as the precuneus, cuneus, caudate, and cerebellar regions, was associated with better performance on CVLT measures, including learning, short- and long-delay recall, and semantic clustering. Functional connectivity from both hippocampi to primarily right prefrontal regions during verbal encoding associated with better CVLT performance. In-scanner word recognition accuracy was more strongly associated with activation of parietal and occipital regions, and with functional connectivity between the right hippocampus and bilateral parietal and temporal regions. Our findings characterize the patterns underlying verbal memory abilities in midlife postmenopausal women. The patterns identified here may act as a foundation for better interpreting the effects of hormonal changes and menopausal symptoms on cognition at midlife, and for identifying neural targets for pharmacological and lifestyle interventions aimed at sustaining womens memory function.

Numerical study of the size-based, shear-induced separation ofcirculating tumour cells from white blood cells in liquid biopsies

Owen, B. — 2025-10-14

Circulating tumour cells (CTCs) are promising biomarkers for early cancer detection, yet their extreme rarity in blood necessitates efficient separation from white blood cells (WBCs) in lysed liquid biopsies. Inertial microfluidics offers a high-throughput, label-free approach to this challenge by leveraging size-dependent lateral migration. However, experimental observations reveal that WBCs migrate more rapidly than predicted, reducing separation performance. Using 3D lattice-Boltzmann-immersed-boundary-finite-element simulations, we characterized the migration dynamics of CTCs and WBCs in a straight microchannel. Our results reveal that the presence of a CTC enhances WBC cross-streamline migration, providing a mechanistic explanation for WBC contamination in CTC-enriched outlets. The numerical model capturing heterochiral orbital dynamics was validated experimentally, confirming the role of intercellular hydrodynamic interactions. These findings underscore the critical role of intercellular interactions in inertial microfluidic systems and provide guidance for optimizing suspension concentration and channel geometry to improve purity in rare cell isolation.

Age differences in electrocortical dynamics during uneven terrain walking

Liu, C. — 2025-10-14

Walking on uneven terrain becomes more difficult as we age, and gait becomes less automatic. Using mobile brain imaging via high-density electroencephalography (EEG) can provide insight into the neural mechanisms contributing to reduced mobility capability with aging. The objective of this study was to quantify age differences in electrocortical dynamics during uneven terrain walking, both averaged across many strides and variations within a stride. We included 31 young adults and 71 older adults for analysis. All participants walked on an uneven terrain treadmill with four levels of terrain difficulty at their self-selected speed. Compared to younger adults, older adults exhibited a greater increase in step duration variability and mediolateral sacral excursion variability as the terrain became more uneven. We identified multiple brain regions involved during walking on uneven terrain. Regardless of age group, walking on uneven terrain compared to flat terrain led to a widespread change of electrocortical dynamics in the brain, especially in the alpha (8-13Hz) and beta (13-30Hz) band power. In the parieto-occipital region, younger adults experienced a greater reduction in alpha and beta power with increasing terrain unevenness compared to older adults. We also assessed how intra-stride power fluctuations changed with terrain unevenness and age group. Greater intra-stride power spectral fluctuations in the occipital area were associated with greater terrain unevenness for younger adults, but not for older adults. In summary, older adults showed a greater increase in gait variability than younger adults as the terrain became more uneven, but exhibited a lack of modulation of parieto-occipital activity in response to terrain unevenness. The lack of task-related power modulation may suggest reduced cortical network flexibility in older adults. The absence of increased parieto-occipital activity when walking on uneven versus flat surfaces in older adults may also indicate that, unlike younger adults, older adults already heavily rely on visual processes during flat surface walking and may therefore have reduced occipital modulation range remaining to cope with the visuomotor processing demands of walking on uneven surfaces.

Xenophagocytosis blockade enhances interspecies chimerism

Wang, S. — 2025-10-15

Organ shortage remains a major challenge in transplantation medicine. Interspecies blastocyst complementation is a promising approach to generate human organs in livestock hosts. However, getting xenogeneic donor cells to engraft and expand at early stages remains challenging. Here we identify an innate immune barrier, wherein host macrophages selectively recognize and eliminate viable xenogeneic donor cells. These events represent a form of phagoptosis and highlight a xenogeneic clearance process that we term xenophagocytosis. We identify the mechanism by which host macrophages selectively phagocytize xenogeneic donor cells: xenogeneic cells display elevated phosphatidylserine, an "eat-me" signal recognized by host macrophages through phagocytic receptor Axl. Xenophagocytosis blockade improves both rat and human donor chimerism in mouse embryos, indicating a conserved mechanism. These findings reveal potential mechanisms by which innate immune cells eliminate xenogeneic cells in early embryogenesis to preserve species integrity and offer improved strategies for generating human organs in livestock.

IDBac: an open-access web platform and compendium for the identification of bacteria by MALDI-TOF mass spectrometry.

Krull, N. K. — 2025-10-15

The identification and analysis of bacteria is central to the microbiological sciences. While gene sequencing methods have been the standard to achieve this, use of MALDI-TOF mass spectrometry (MS), particularly in clinical microbiology, provides high-throughput identification to the subspecies level. However, biotyping has yet to be adopted outside of clinical settings due to the lack of a centralized public database of MS protein signatures that would facilitate isolate identification via spectral comparison. Further, current platforms lack meaningful ways to compare multiple properties from large numbers of bacterial isolates. Herein we present the IDBac web platform, a crowd-sourced central knowledgebase of protein MS signatures of >1400 strains spanning 6 bacterial phyla. Accompanying the knowledgebase is analysis infrastructure to identify unknown isolates, probe relationships within culture collections using metadata integration, and visualize specialized metabolite differences within groups of closely related bacteria. To highlight this utility and encourage wide community contribution, examples of each are presented.

Standardizing the mouse MCAO Stroke Model: Distinguishing Internal Carotid vs Pterygopalatine Artery Occlusion

Isbatan, A. — 2025-10-21

BackgroundThe intraluminal middle cerebral artery occlusion (MCAO) model is widely used in preclinical stroke research, but outcomes remain inconsistent because of variable occlusion times and inadvertent filament placement into the pterygopalatine artery (PPA). MethodsWe applied a modified version of the Koizumi method to directly visualize the internal carotid artery (ICA)-PPA bifurcation and systematically compare targeted ICA versus PPA occlusion. We combined multiple complementary methods - survival studies, neurological scoring, 2,3,5-Triphenyltetrazolium chloride (TTC) infarct staining, Evans blue blood brain barrier (BBB) permeability assays, laser speckle cerebral blood flow imaging, small-animal magnetic resonance imaging (MRI), and evaluation of inflammatory gene expression through qPCR - to rigorously characterize injury patterns, survival, and inflammatory responses. ResultsICA occlusion approximately caused a rapid 70% reduction in cerebral blood flow, progressive infarction (15 min: smaller infarct with approximately 50% 7-day survival; 30 min: moderate infarct but >50% mortality), and cortical edema. In contrast, PPA occlusion produced only a gradual 44% decline in blood flow, small infarcts at 60 min) and preserved survival (80% at 7 days). Finally, magnetic resonance imaging in 15-minute ICA occluded mice identified the development of cortical edema which was not observed in sham, 15-minute PPA, or 60-minute PPA mice. ConclusionThese findings identify 15-minute ICA occlusion as the most practical balance between reproducible infarction and survival for long-term studies, while 30 minutes is best reserved for short-term infarct induction when survival is less critical. By resolving two critical sources of MCAO variability, occlusion duration and vascular targeting, this study provides a standardized protocol and evidence-based occlusion benchmarks that will improve reproducibility in preclinical stroke studies

Unraveling Streptococcus pyogenes Carriage: Genomic and Transcriptomic Insights from Acute and Post-Treatment Phases

Faiola, N. — 2025-10-22

The carrier state of Streptococcus pyogenes (Group A Streptococcus, GAS), is defined by the presence of the organism without symptoms or an immune response. Carriage poses challenges for clinical management and contributes to transmission in the community. Prior studies suggest genetic and phenotypic differences between isolates from symptomatic (acute) infections and those from carriers, yet comprehensive genomic and transcriptomic analyses of naturally acquired human GAS carriage have been lacking. In this study, we collected longitudinal samples from individuals before and after antibiotic treatment for acute pharyngitis, performing whole genome sequencing and RNA-seq on selected isolates. This is the first report of transcriptional profiling of GAS directly from human derived oropharyngeal swabs. Genomic analysis revealed no consistent carrier-specific genotypes, and in vitro assays showed no major differences in biofilm formation or antibiotic susceptibility between carrier and acute isolates. Transcriptional profiling from oropharyngeal swabs identified distinct gene expression patterns when comparing the acute infection and post-treatment carriage phases, although early acute expression profiles did not predict treatment failure. These findings suggest that GAS carriage is likely influenced by conserved bacterial traits as well as host factors, advancing our understanding of GAS persistence and transmission. SummaryLongitudinal samples were collected from individuals before and after treatment for acute Group A Streptococcal pharyngitis. Whole genome sequencing and RNA-seq was performed on selected isolates. This is the first report of transcriptional profiling of GAS directly from human oropharyngeal swabs. Distinct gene expression patterns were observed when comparing the acute infection and post-treatment carriage.

NicheProt: Cell-type-resolved proteomics of tissue compartments

Wu, Y.-C. — 2025-10-24

Spatial proteomics uncovers the molecular underpinnings of cellular function in intact tissues. Laser capture microdissection coupled with mass spectrometry enables comprehensive proteomic profiling of selected tissue regions, but typically does not support cell-type-specific proteomic analysis. We present NicheProt, a 3D optical microscopy-guided, photobleaching-mediated cell barcoding approach for isolating intact specific cell types from defined microanatomical tissue compartments or niches. Using sequential bottom-up proteomic analysis, we defined two distinct phenotypes of CD11c dendritic cells based on their spatial locations in the inflamed mouse spleen. These two compartment-specific dendritic cell populations were characterized by proteomic signatures differing in the levels of 54 proteins. This 3D tissue microscopy-guided method offers cell-type and microregion-resolved proteomic analysis, facilitating the proteomic discovery of previously unrecognized cell subtypes and their functional roles in distinct tissue compartments.

Integrated metabolomic and genomic insights into amino acid incorporation within the hybrid polyketide-alkaloid antibiotic TLN-05220

Cordoza, J. L. — 2025-10-31

Actinobacteria are a rich source of bioactive compounds and unique biosynthetic chemistry. Micromonospora echinospora subsp. challisensis NRRL 12255 produces the aromatic polyketide TLN-05220, which exhibits nanomolar activity against antibiotic-resistant human pathogens including vancomycin-resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus. The pentangular polyphenol core of TLN-05220 is decorated with a piperazinone moiety, yet the enzymes responsible for the construction of this uncommon modification from amino acid precursors are unknown. Synthetic piperazinone-containing molecules have diverse antimicrobial, antiviral, anticancer, and anti-inflammatory bioactivity profiles, and determining biosynthetic routes for the assembly of this heterocycle may enhance drug discovery and medicinal chemistry efforts. We identified a putative TLN-05220 biosynthetic gene cluster (BGC) in the commercially available strain M. echinospora ATCC 15837 containing both type-I and type-II polyketide synthases, two predicted asparagine synthetase-like enzymes, and two genes (tln1 and tln5) that putatively encode pyridoxal 5-phosphate (PLP)-dependent amino acid synthases. Stable isotopic feeding studies coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), identified L-alanine, L-serine, and glycine as metabolic precursors of TLN-05220. Subsequent in vitro enzymology established that Tln1 is a PLP-dependent alanine racemase, while Tln5 performs a stereoselective {beta}-substitution reaction of O-phospho-L-serine with a preferential D-alanine nucleophile. Alanine racemization and Tln5 pseudodipeptide L-serine-C{beta}-N-D-alanine (D,L-PDP) incorporation into TLN-05220 were further supported using deuterated intermediates and mass spectrometry techniques. Establishing the enzymes that catalyze amino acid installation within TLN-05220 inspires further biosynthetic discovery and engineering while enabling the biocatalytic syntheses of novel amino acid-containing polyketide antibiotics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/685158v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@4402e8org.highwire.dtl.DTLVardef@11af789org.highwire.dtl.DTLVardef@1717c5borg.highwire.dtl.DTLVardef@19b6529_HPS_FORMAT_FIGEXP M_FIG C_FIG

Private QTLs and the Genetic Architecture of Hierarchical Size Traits: From Body Size to Sex-Specific Plasticity

Vea, I. M. — 2025-10-31

Sex-specific size plasticity (SSP) is the phenomenon whereby the size of one sex is more environmentally sensitive than the other, and is thought to underlie the developmental regulation and evolution of sexual size dimorphism (SSD). Sex-specific plasticity is a higher order phenotype that emerges due to the effect of the environment and sex on core growth regulatory mechanisms. Genetic variation in SSP necessarily requires sex- and environment-specific variation in growth, yet the developmental-genetic mechanisms enabling such context-dependent size variation remain poorly understood. Using a genome-wide association study (GWAS) and functional validation in Drosophila melanogaster, we dissected the genetic architecture of body size, size plasticity, SSD, and SSP across 196 isogenic lineages. We find that each phenotype is governed by largely non-overlapping sets of loci, with most candidate variants lying outside canonical growth pathways. Instead, size trait are shaped by "private QTLs", whose effects are limited to specific sex, trait, or environmental contexts. Functional knockdown of selected candidate genes for SSP revealed that while most did not affect SSP directly, many influenced body size, SSD, and size plasticity, in a manner consistent with their nested phenotypic relationships. Together, our results suggest that context-dependent alleles in genes peripheral to core growth regulatory pathways drive variation in SSD and SSP, offering a mechanistic explanation for their evolutionary lability and highlighting the role of private QTLs in structuring complex trait architecture. Significance StatementSexual size dimorphism (SSD), the differences in body size between females and males, is highly variable among species and yet the developmental genetic mechanisms that generate it and drive its evolution remain elusive. Our work reveals a modular genetic architecture underlying SSD in Drosophila melanogaster, showing that variation in body size, its sex-specific plasticity, and the resulting SSD are controlled by largely non-overlapping, context-dependent loci. This finding provides critical insight into how complex morphological differences between the sexes are genetically organized, allowing for the flexible and independent evolution of hierarchical traits.

A Chromatin-Structure-Guided Framework for Predictive and Interpretable Regulatory Genomics

Ye, B. — 2025-11-05

Chromatin organization shapes gene regulation by linking distal elements across megabase scales, yet most predictive genomics models still treat the genome as linear, without incorporating three-dimensional structure. Hi-C provides genome-wide chromatin conformation information, but its contact maps are population-averaged, distance-biased, and noisy, obscuring the biologically specific contacts. We present CHROME, a framework built on a self-avoiding polymer ensemble null model that identifies physically specific, non-random Hi-C contacts. By integrating these contacts into graph representations, CHROME enables efficient information transfer across spatially connected loci. It integrates sequence, chromatin accessibility, or pre-trained embeddings into a graph attention architecture to predict cell line-specific ChIP-seq profiles, consistently outperforming local encoder baselines and generalizing to an unseen cell line. The resulting graph embeddings also enhance prediction on tissue-specific eQTL and ClinVar variant pathogenicity, outperforming local sequence-based embeddings. Beyond predictive performance, CHROME provides interpretability through attention-derived neighbor-to-center contributions that reveal how spatially connected loci influence local regulatory activity over multi-megabase distances. Together, these results show that incorporating physically validated chromatin interactions enables more accurate and interpretable modeling of gene regulation and variant effects.

Streptococcus pneumoniae fratricide-induced cell lysis does not require the type IV competence pilus.

Borowska, A. K. — 2025-11-06

Streptococcus pneumoniae, a bacterium that becomes competent naturally, can undergo transformation and fratricide during which different genes, including antibiotic-resistant ones, can be transferred. A Type IV pilus (T4P) is involved in the DNA take up during transformation or fratricide-induced (fratricide = same species cell killing) gene transfer. Here, contrary to our expectations, we show that competence T4P is not required in fratricide-induced cell lysis. We have utilized pilus and pilus- attackers, and a {beta}-galactosidase+ ({beta}-gal+) victim for our experiment. Transformation, gene transfer efficiency, and cell lysis measured by release of {beta}-gal+ were tested at different optical cell densities (OD) under competence-induced (+CSP) or non-competence (-CSP) conditions. We have optimized experiment conditions to observe cell lysis, which only occurred in the media that do not contain choline (inhibits cell lysis). As expected, we have observed transformation and gene transfer with the pilus+ strain, but none of those two processes with the pilus- strain. In the case of {beta}-gal release during fratricide, we have observed {beta}-gal activity in both cases whenever the pilus+ or pilus- strain was mixed with the {beta}-gal+ victim. Our data indicate that type IV competence pilus, while crucial for DNA take up during transformation fratricide, is not needed during cell lysis.

TriLeukeVax: A CD80/IL-15/IL-15Rα Expressing Autologous AML Cell Vaccine Elicits Robust Anti-Leukemic Cytolytic Activity

Du, J. — 2025-11-09

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is associated with poor outcomes due to frequent relapse after remission induction. While hematopoietic stem cell transplantation (HSCT) can improve survival, many individuals, especially older patients, are ineligible. Prior immunotherapies have not reliably induced effective anti-leukemic immunity and have been associated with severe and unpredictable toxicities. Thus, there is a need for safe and effective therapies that reduce relapse and increase overall survival (OS). We have developed a universally applicable, patient-specific, lentivirally engineered autologous AML cell vaccine, TriLeukeVax (TLV), designed to stimulate leukemia-specific cytolytic immune responses in AML patients in remission. To generate TLV, AML cells are engineered to express the highly synergistic combination of the co-stimulatory protein CD80 and the IL-15/IL-15-receptor alpha (IL-15R) heterodimer. Prior proof-of-concept (POC) studies demonstrated eradication of disease in >80% of leukemic mice with serial administration of TLV. In the current studies, TLV was generated from 59/60 cryopreserved, diagnostic bone marrow-derived patient AML samples. Ex vivo priming of post-remission patient T-cells by ex vivo co-culture with autologous TLV stimulated robust proliferative and cytotoxic responses. In secondary co-cultures, T-cells previously primed by initial co-culture with TLV, showed greater clonal expansion and leukemia-specific cytolytic activity towards de novo autologous AML blasts than did control, unprimed T-cells. The enhanced anti-leukemic activity of TLV-primed T-cells against de novo AML confirms the potential for vaccine administration to effectively target minimal residual disease (MRD) persisting after chemotherapy and reduce relapse. Key PointsO_LITriLeukeVax induces proliferation, activation, and effective anti-leukemic cytolytic responses in remission T-cells. C_LIO_LIPrimed T-cells show polyclonal expansion and transcription profiles associated with proliferation, memory, and cytotoxicity. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/687000v2_ufig1.gif" ALT="Figure UFIG"> View larger version (38K): org.highwire.dtl.DTLVardef@5c1827org.highwire.dtl.DTLVardef@35b2e2org.highwire.dtl.DTLVardef@1732914org.highwire.dtl.DTLVardef@18df655_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOVisual Abstract.C_FLOATNO TriLeukeVax (TLV) workflow and mechanism of action. Diagnostic AML patient bone marrow aspirates are collected, purified in Ficoll and cryopreserved. Samples are thawed and lentivirally transduced to produce TLV. Vaccination of AML patients in remission with TLV will stimulate the activation and expansion of leukemia-specific T-cells, effector memory cells, and NK cells by combining the co-stimulatory effects of CD80 with immune stimulation by the IL-15/IL-15R heterodimer expressed by the transduced AML cells, thereby targeting MRD and potentially increasing relapse-free survival in AML patients. C_FIG

From Peaks to Patterns

Fathollahian, H. — 2025-11-10

MRS is a non-invasive method for gathering information about cellular metabolism and can be used to study neurological and metabolic disorders. However, the clinical utility is limited by low signal quality, overlapping spectral peaks, and the inability to interpret and compare metabolite data. We developed an interactive visual analysis system that integrates methods for filtering, quality measurement, and comparative summaries of MRS signals, enabling researchers to more effectively evaluate and interpret spectral data. This will enable researchers to conduct a more accurate evaluation of spectral data, thereby facilitating a deeper understanding of the molecular processes underlying complex neurological and metabolic processes.

SpaTRACE: Spatiotemporal recurrent auto-encoder for reconstructing signaling and regulatory networks from spatiotemporal transcriptomics data

Zhou, H. — 2025-11-21

Cell-cell communication (CCC) drives the coordinated cellular dynamics underlying development, regeneration, and disease. Recent advances in spatiotemporal transcriptomics now enable measurement of gene expression within both spatial context and developmental progression. However, most existing CCC methods depend heavily on curated ligand-receptor (LR) databases and assume steady-state expression, limiting their applicability to understudied species and preventing robust inference of dynamic signaling cascades. Here, we introduce SpaTRACE, a transformer-based Granger-style recurrent autoencoder for de novo reconstruction of signaling and gene-regulatory interactions directly from developmental spatiotemporal transcriptomics data. SpaTRACE jointly embeds LR pairs, transcription factors (TFs), and target genes (TGs) into a unified latent space that integrates spatial neighborhoods with temporal causality. Trained to predict future TG expression along pseudotime-sampled trajectories, the model learns biologically meaningful embeddings that capture LR[->]TG signaling and TF[->]TG regulatory influence without relying on pathway priors. Across synthetic datasets, SpaTRACE accurately reconstructs LR-TG interactions, TF-TG regulation, and correct LR pairings, outperforming existing CCC tools--especially under pathway-agnostic settings. Applied to mouse midbrain development and axolotl brain regeneration, SpaTRACE recovers canonical signaling modules, identifies stage-specific transitions, and uncovers previously under-characterized interactions, producing CCC signals with high co-expression fractions and bivariate Morans I comparable to leading spatial methods. Together, SpaTRACE establishes a general, statistically powerful framework for dissecting dynamic intercellular communication and regulatory networks from spatiotemporal transcriptomics. A user-friendly implementation is available at: https://github.com/VariaanZhou/SpaTRACE.git.

Acute immune modulation with poly-salicylic acid particles ameliorates pain and structural damage in post-traumatic osteoarthritis

Felder, M. L. — 2025-11-24

Joint inflammation is a hallmark of post-traumatic osteoarthritis (PTOA) progression and a recognized driver of articular destruction and symptoms. Despite its known pathological role, inflammation has not been successfully targeted to treat PTOA. With the hypothesis that blocking the acute influx of systemically-derived immune cells can mitigate injury-induced inflammation and downstream PTOA disease severity, we targeted immune cell recruitment via systemically-administered poly salicylic acid (PolySA) particles. This formulation targets immune cells in circulation, namely neutrophils and monocytes, to inhibit their vascular extravasation into injured tissue. Employing a murine joint injury model, we show that PolySA particles reduced neutrophil and monocyte recruitment to the synovium by >50% when administered acutely after injury. Sex-specific therapeutic effects of PolySA emerged 7d post-ACLR, whereby female knee joints exhibited increased cathepsin activity and alleviation of knee hyperalgesia. Despite also observing reduced immune cell recruitment in male mice treated with PolySA, therapeutic effects were entirely absent in males. RNAseq of female synovium revealed a transcriptomic signature indicative of accelerated immune resolution and matrix remodeling in PolySA-treated female mice. Analyses at a timepoint of established disease showed that PolySA-treated female mice exhibited sustained pain alleviation, reduced osteophyte formation, and decreased histopathological PTOA and synovitis severity scores. Together, these findings indicate that blocking acutely-recruited immune cells to the local joint microenvironment via systemic PolySA particle treatment is a promising therapeutic for PTOA prevention by reprogramming early injury-induced inflammation.

In vitro design of intrathecal drug administration therapies

Ayansiji, A. — 2025-11-29

1Due to the scarcity of reliable in vivo data, the pharmacokinetics of intrathecally (IT) administered drugs remain inadequately quantified. Designing new therapies is further hindered by variability in experimental methods, inter-individual and inter-species differences, and poor reproducibility across animal and human studies. To address these limitations, we developed an anatomically accurate, subject-specific replica of the cerebrospinal fluid-filled spaces of the human central nervous system (CNS) using a multistep mold/casting process. The 3D-printed, transparent, deformable CNS phantom enables precise control of infusion and physiological parameters, allowing systematic generation of reliable and repeatable biodispersion data for lumbar IT infusion protocols. Pulsatile artificial cerebrospinal fluid (CSF) flow within the closed system was tuned to replicate subject-specific stroke volumes and flow rates observed in MRI. The models optical clarity facilitated high-speed visualization and tracking of tracer dispersion, exceeding the temporal resolution of current neuroimaging techniques. Experimental series spanning physiologically relevant CSF and infusion conditions enabled quantification of the spatiotemporal distribution of IT-administered tracers. Inversion of the parabolic diffusion equation provided estimates of the coefficient of effective dispersion. A distributed pharmacokinetic model was used to evaluate the influence of chemical kinetics and mass transfer on tracer behavior. The proposed experimental apparatus for in vitro design of IT therapies offers a complementary or alternative approach to traditional trial-and-error animal studies.

Disrupted tRNA modification leads to intestinal mitochondrial dysfunction and microbial dysbiosis

Ran, D. — 2025-12-01

Background and aimsTransfer RNA (tRNA) modifications determine translation fidelity and efficiency. It occurs through the action of specific enzymes that modify the nucleotides within the tRNA molecule. Our previous study demonstrated tRNA modopathies and altered queuine-related metabolites in inflammatory bowel diseases. Queuine tRNA-ribosyltransferase catalytic subunit 1 (QTRT1) and QTRT 2 co-localize in mitochondria and form a heterodimeric TGT participating in tRNA Queuosine (tRNA-Q) modification. Human body acquires Queuine/Vitamin Q from intestinal microbiota or from diet. However, the roles of tRNA-Q modifications in the maintenance of intestinal mitochondrial homeostasis and microbiome are still unclear. MethodsWe used publicly available human IBD datasets, QTRT1 knockout (KO) mice, QTRT1 intestinal epithelial conditional KO (QTRT1{Delta}IEC) mice, cultured cell lines with QTRT1-specific siRNA, and organoids from patients with IBD to investigate the mechanism of tRNA-Q modifications in intestinal mitochondrial homeostasis and therapeutic potential in anti-inflammation. ResultsIn single cell RNA sequencing datasets of human IBD, we identified significant reduced intestinal epithelial QTRT1 expression in the patients with Crohns Disease. Using publicly available datasets, we identified significantly changes of Vitamin Q-associated bacteria in human IBD, compared to the healthy control. Qtrt1-/- mice had significant reduction of Q-associated bacteria, e.g., Bacteroides. Alcian Blue and Mucin-2 staining revealed mucosal barrier damage and disrupted homeostasis, with reduced colonic cell proliferation. Intestinal tight junction integrity was impaired in QTRT1-KO mice, as evidenced by reduced ZO-1 and increased Claudin-10 expression. QTRT1{Delta}IEC mice also showed dysbiosis and disrupted TJs. ATP synthesis was significantly decreased in the colon of QTRT1-KO mice, accompanied by severe mitochondrial dysfunction: reduced mitochondrial quality, Cytochrome-C release, and mitochondrial DNA (mtDNA) leakage. Mitochondrial dysfunction contributed to colonic cell death, as shown by elevated expressions of Cleaved Caspase-3 and Cleaved Caspase-1, increased BAX/Bcl-2 ratio, and positive TUNEL signals. Elevated CDC42, CD14, and CD4 levels in QTRT1-KO colon suggested mucosal immune activation and tissue repair responses. QTRT1-deficient CaCO2-BBE cells showed mitochondrial dysfunction. Cytochrome-C and mito-DNA release leading to cell death characterized by elevated expressions of Cleaved Caspase-3 and Caspase-1, increased BAX/Bcl-2 ratio, and higher apoptosis rate. Organoids isolated from patients with IBD showed reduced levels of QTRT1 and dysfunctional mitochondria. Restoring mitochondrial function leads to enhanced QTRT1. ConclusionsThese findings underscore the critical role of QTRT1/Q-tRNA modification in maintaining intestinal and microbial homeostasis. Mechanistically, QTRT1 loss impacts mitochondrial integrity and mucosal homeostasis. Our study highlights the novel roles of tRNA-Q modification in maintaining mucosal barriers and innate immunity in intestinal health. What is already known about this subject?O_LIEukaryotes acquire queuine (q), also known as Vitamin Q, as a micronutrient factor from intestinal microbiota or from diet. C_LIO_LIVitamin Q is needed for queuosine (Q) modification of tRNAs for the protein translation rate and fidelity. C_LIO_LIQueuine tRNA-ribosyltransferase catalytic subunit 1 (QTRT1) is reduced in human IBD. C_LIO_LIHowever, health consequences of disturbed availability of queuine and altered Q-tRNA modification in digestive diseases remain to be investigated. C_LI What are the new findings?O_LIQTRT1 deficiency leads to altered microbiome and reduced Vitamin Q-associated bacteria in human IBD and a QTRT1 KO animal model. C_LIO_LIQTRT1 protects the host against losing intestinal integrity during inflammation. C_LIO_LIQTRT1 localizes in mitochondria and plays novel functions by maintaining intestinal mitochondrial function. QTRT1 loss impacts tRNA modification in the intestine, linking to mitochondrial integrity and mucosal homeostasis. C_LIO_LIHuman IBD showed reduced levels of QTRT1 and dysfunctional mitochondria. Restoring mitochondrial function leads to enhanced QTRT1. C_LI How might it impact on clinical practice in the foreseeable future?Targeting tRNA-Q modification in enhancing mitochondrial function will be a novel method to maintain intestinal health.

B7-H3 upregulation in ischemic stroke: friend or foe?

Challa, S. R. — 2025-12-02

B7-H3 (CD276) is an immune checkpoint co-signaling molecule expressed on immune and non-immune cells. It is best known for suppressing T-cell responses but can also promote inflammation depending on the microenvironment. In neuroinflammatory models such as experimental autoimmune encephalomyelitis, B7H3 expression increases concomitantly with the inflammatory response, and its inhibition is associated with reduced disease progression. Although its role in ischemic stroke remains unclear, we hypothesized that cerebral ischemia/reperfusion (I/R) would upregulate B7-H3 expression in the ischemic brain and that increased B7-H3 expression would positively correlate with pro-inflammatory cytokine expression. Young and aged male and female rodents, including normotensive and spontaneously hypertensive rats to model comorbid hypertension, underwent transient middle cerebral artery occlusion (MCAO) followed by reperfusion. Brain tissue was collected on post-ischemic days 1, 3, 5, or 7. B7-H3 mRNA was analyzed by real-time PCR, whereas protein expression was assessed by Western blotting and immunohistochemistry at selected time points. B7-H3 expression was significantly upregulated in the ischemic brain across sexes, age groups, and species. The extent of B7-H3 degradation in the ischemic brain was influenced by species, sex, age, and time after cerebral I/R. Upregulation of B7-H3 was observed at both the mRNA and protein levels, and increased expression was localized primarily to the somatosensory cortex and caudate putamen in the ipsilateral hemisphere, the main regions affected in this MCAO model. Elevated B7-H3 expression in the ischemic brain positively correlated with the pro-inflammatory mediator TNF. The temporal profile of B7-H3 expression observed in rats paralleled the early inflammatory phase associated with secondary tissue damage following ischemic stroke. These findings identify B7-H3 as an ischemia-induced immune checkpoint molecule in the brain that may modulate post-stroke immune responses and support further investigation into its beneficial versus detrimental roles in neuroinflammation, as well as its potential as a therapeutic target following cerebral I/R.

Tiarins, a diverse family of natural Trojan-horse aminoacyl-tRNA synthetase inhibitors discovered by genome mining

Travin, D. Y. — 2025-12-02

Trifolitoxin (TFX) is a ribosomally synthesized and post-translationally modified peptide antibiotic produced by Rhizobium anhuiense T24. Although discovered more than half a century ago, its mechanism of action has remained elusive. Here we demonstrate that TFX inhibits arginyl-tRNA synthetase (ArgRS), an essential translation enzyme. TFX acts as a Trojan-horse antibiotic: it enters cells via the inner membrane oligopeptide transporter YejABEF and then undergoes partial proteolysis by the aminopeptidase PepN to release a "warhead" that binds ArgRS and arrests translation. A systematic analysis of prokaryotic genomes revealed that TFX belongs to a widespread family of modified peptides that we designate the tiarins. In addition to ArgRS inhibitors, the tiarin family includes compounds that specifically target at least seven other aminoacyl-tRNA synthetases. These findings define a previously unrecognized family of natural antibiotics and provide a framework for developing a new class of inhibitors targeting various tRNA synthetases.

Early blood-spinal cord barrier stabilization preserves peri-lesional tissue and improves recovery after SCI

Chen, C. — 2025-12-07

Vascular disruption is an early and critical event in spinal cord injury (SCI), but its contribution remains poorly understood. Using novel in vivo two-photon dual-dye imaging, we found simultaneous blood-spinal cord barrier (BSCB) leakage and venous dilation in both the injury epicenter and adjacent transitional segment after cervical SCI in rats. Notably, vascular permeability in the transitional zone preceded axonal and neuronal loss, revealing a therapeutic window. Systemic delivery of ferulic acid-glycol chitosan (FA-GC) nanoparticles, a membrane-sealant, rapidly stabilized the compromised vasculature, reduced neuronal loss in the transitional region, and improved forelimb muscle strength. These findings identified acute vascular leakage beyond the injury epicenter as a driver of secondary pathology and highlight early vascular stabilization as promising therapeutic strategy.

Presynaptic GABAB autoreceptors suppress neurotransmitter release during repetitive stimulation via the Gβγ-SNARE pathway.

Zurawski, Z. — 2025-12-13

GABAergic signaling provides the brains primary inhibitory mechanism with defects linked to epilepsy, anxiety, depression, insomnia, schizophrenia and neurodegeneration. A key regulatory mechanism is autoinhibition of GABA release during repetitive activity via presynaptic Gi/o-coupled GABAB receptors, supporting synaptic tuning and memory formation, and limiting neurotransmitter spillover. Exogenous GABAB receptor agonists reduce presynaptic Ca2+ entry by inhibiting calcium channels. However, using transgenic mice expressing a mutant SNAP25 with diminished ability to bind G{beta}{gamma} (SNAP25{Delta}3), we show that suppression by GABAB autoreceptors requires intact G{beta}{gamma}-SNARE interactions. Imaging of presynaptic Ca2+ transients in GABAergic axons showed no GABA-mediated autoreceptor suppression of Ca2+ entry during stimulus trains. In contrast, application of the exogenous GABAB receptor agonist baclofen profoundly inhibited Ca2+ entry, which could be partially reversed by exogenously elevating cAMP, indicating a complementary role of inhibition of adenylyl cyclase. Baclofen reduced spontaneous IPSC frequency and amplitude and both effects were diminished in SNAP25{Delta}3 mice, consistent with inhibition at Ca2+ channels and SNARE complexes. Physiological GABA-mediated and exogenous GABAB receptor activation thus produce distinct outcomes on GABAergic neurotransmission, indicating that synthetic drug application to neurons does not faithfully recapitulate endogenous signaling pathways. We conclude that endogenous rapid GABAB autoreceptor signaling inhibits neurotransmitter release primarily by G{beta}{gamma}-mediated inhibitions of SNARE mechanisms, whereas prolonged agonist application additionally suppresses Ca2+ influx via cAMP signaling.

MALDI-FISH for co-localization of brominated metabolites and Pseudovibrio spp. in Aplysina tissue

McAtamney, A. — 2025-12-23

Bromotyrosine-containing natural products have long been isolated from marine sponges of the order Verongiida. Recent studies have questioned the source of these natural products whether production occurs through the sponge, its diverse microbiota, or perhaps a combination of both. While studying the uptake of engineered bacterial strains by sponges, we have observed production of fistularin-3 in sponge. To explore these results further, we employed multimodal imaging techniques to co-localize microbial metabolites with fluorescent in situ hybridization (FISH) probes. Here, we built on previously developed MALDI-FISH methods to add to the conversation on brominated metabolite production in marine sponges. Using MALDI-MSI, we measured fistularin-3, a poly-brominated natural product previously isolated from Aplysina aerophoba, present in our A. aerophoba samples that were inoculated with Pseudovibrio brasiliensis. A recent study reported that P. brasiliensis produced fistularin-3 in pure culture, but this data has not yet been reproduced. We employed MALDI-MSI and FISH on the same sponge cryosections to colocalize the spatial distribution of fistularin-3 with P. brasiliensis in sponge tissue. Despite the caveats of this study, our data suggests that perhaps both sponge and microbe may be required for production of fistularin-3.

Backbone Hydrogen Bonding as a Determinant of Condensate Material States

Zhang, Y. — 2025-12-23

Condensate material states ranging from liquid droplets to gels have direct functional consequences, but their molecular determinants, especially for gels, are poorly understood. Here we combined optical microscopy, NMR spectroscopy, and molecular dynamics (MD) simulations to test the hypothesis that backbone hydrogen bonding is a determinant for the material states of XYssYX tetrapeptides. IAssAI forms droplets but AIssIA forms gels; MD simulations show a higher level of hydrogen bonds in AIssIA condensates than in IAssAI condensates. Addition of a low-hydrogen-bonding peptide, AAssAA, converts AIssIA gels into droplets. The same effect is achieved by methylating the backbone amides of AIssIA to block hydrogen bonding. 1H-1H nuclear Overhauser effect spectroscopy, coupled with MD simulations, reveals strong, backbone hydrogen bonding-buttressed molecular networks in AIssIA gels. These results demonstrate that, in addition to sidechain-sidechain interaction strength, backbone hydrogen bonding, by promoting directional growth, is a determinant of condensate material states.

Differential Cerebral White Matter Tract Alterations in Generalized Anxiety Disorder Revealed by Ultra-High Field 7T Diffusion-Weighted Imaging

Chen, C.-L. — 2025-12-27

BackgroundGeneralized anxiety disorder (GAD) is characterized by chronic worry and emotional dysregulation, yet its underlying white matter (WM) architecture remains inconsistent in previous neuroimaging studies. This study aimed to delineate microstructural WM alterations in GAD using ultra-high field (7T) diffusion tensor imaging (DTI) and advanced correlational tractography, evaluating their associations with symptom severity and diagnostic-aided value. MethodsEighty-eight participants (27 GAD, 61 healthy controls, HCs) underwent 7T DTI scanning with 1.5-mm isotropic resolution. Whole-brain correlational tractography was applied to identify tracts exhibiting significant group-related differences in diffusion indices while controlling for demographic covariates. Associations with Hamilton Anxiety Rating Scale (HAM-A) and Penn State Worry Questionnaire (PSWQ) scores were examined, and machine learning-based models were used to assess the diagnostic-aided utility of identified WM features. ResultsTwo tract bundles showed significant fractional anisotropy (FA) alterations in GAD: decreased FA in the right prefrontal pathway (PFDR = 0.039) and increased FA in the right cingulum (PFDR < 0.001). The anterior portions of both tracts exhibited stronger effects of GAD. FA in the right cingulum positively correlated with HAM-A and PSWQ scores, indicating that greater microstructural coherence was associated with higher anxiety and worry severity (both PFDRs < 0.001). Inclusion of WM features could improve classification performance beyond neuropsychological measures alone. ConclusionsUltra-high field 7T tractometry revealed a differential pattern of WM abnormalities in GAD, suggesting weakened prefrontal control and hyper-integrated cingulate connectivity as structural correlates of emotional dysregulation. These findings indicate the potential of 7T DTI markers for advancing mechanistic and diagnostic understanding of GAD.

Ultrastructural changes at auditory nerve synapses following moderate noise exposure

Gorman, D. — 2025-12-31

Moderate noise exposure is a common experience, yet its impact on central auditory synapses remains poorly understood. We study this issue at the first synapses in the central auditory pathway formed by auditory nerve afferents onto bushy cells in the cochlear nucleus, called endbulbs of Held. Non-traumatic noise exposure alters endbulb properties, decreasing the probability of vesicle release and enlarging the pool of releasable vesicles as assessed using electrophysiological methods and immunolabelling. These changes appear homeostatic, to maintain synaptic efficacy during periods of high activity. To identify structural changes underlying the larger vesicle pool, we used serial blockface electron microscopy of endbulbs from control and noise-exposed mice to quantitatively assess synaptic morphology. We observed no differences in the juxtapositional area between endbulbs and bushy cells, nor in the number or density of active zones and postsynaptic densities. Images of endbulb terminals were significantly darker after noise exposure, indicating an increase in the density of synaptic vesicles. These results suggest that moderate noise exposure induces an activity-dependent increase in presynaptic vesicle numbers, consistent with the observed physiological changes in neurotransmitter release. This work sets the stage for high-resolution studies to quantify docked and reserve vesicles. Significance statementNoise exposure is a fact of everyday life, and it is important to understand how noise affects function in the auditory pathway in the brain to understand the full consequences of noise exposure. Electrophysiological experiments indicate that noise triggers a homeostatic increase in the releasable pool of vesicles at auditory nerve synapses. We examined the cellular basis for this change using serial blockface electron microscopy of auditory nerve synapses with and without noise exposure. We reconstructed a number of bushy cells and their presynaptic auditory nerve terminals. After noise exposure, there was no significant increase in the area of synaptic contact or the number or density of synaptic release sites. There was an increase in the number of vesicles near release sites, which may account for the physiological changes. These results emphasize the importance of detailed anatomical studies to study the effects of noise exposure and thus determine the best mechanistic approach for therapies and treatments of noise-induced hearing loss.

Perturbing H-NS function reveals roles in restricting virulence heterogeneity and pathogen adaptation

McLelland, L. — 2026-01-02

Bacterial pathogens must balance rapid expression of virulence genes in host niches with tight repression when not needed to avoid fitness costs and ensure survival. Integration of virulence gene regulatory networks with the conserved global repressor H-NS was critical to achieve this balance. H-NS-mediated repression of virulence genes in non-inducing environments is essential for maintaining virulence genes and has shaped pathogen evolution. However, the role of H-NS-mediated repression in virulence gene activation and pathogen evolution in virulence-inducing conditions is less clear. For instance, although virulence gene expression is often heterogeneous, whether relief of H-NS repression contributes to this heterogeneity remains unknown. Furthermore, whether H-NS repression shapes pathogen evolution in virulence-inducing environments is unclear. Here, we use a Salmonella strain with reduced H-NS DNA-binding affinity to investigate the role of H-NS in virulence gene expression in individual bacteria and pathogen adaptation. We find that reduced H-NS repression increases the fraction of virulence gene expressing cells without eliminating bimodality, resulting in enhanced epithelial cell infection in vitro. Using experimental evolution, we demonstrate that the hns genotype constrains adaptive mutations and that disabling virulence gene expression is a common path to improved fitness in intracellular-like environments. Our results expand the role of H-NS-mediated repression from silencing virulence genes in non-inducing conditions to regulating heterogeneity in inducing conditions and demonstrate that evolutionary conservation of H-NS constrains adaptive strategies in intracellular-like environments.

Conformational changes upon pore blocker removal reveal conductive states of TMEM16A

Stephens, C. A. — 2026-01-03

TMEM16A is a Ca2+-activated anion channel that provides direct electrical feedback to the plasma membrane in response to intracellular Ca2+. Its conductive state remains unresolved, leaving questions about gating, Cl- permeation, and modulation by Ca2+, depolarization, and lipids. To investigate the open state, we performed molecular dynamics simulations of TMEM16A bound to the putative open-state blocker 1PBC. After inhibitor removal, the putative, pore-lining helix TM4 developed kinks at two sites: an upper site that opens the pore for Cl- permeation, and a deeper site causing constriction. A conserved hydrophobic network between TM3 and TM4 persisted in most open structures but separated during extreme dilation, allowing lipids to transiently block the pore. Patch-clamp recordings indicated that the intact network promotes activation. Further simulations yielded >60 Cl- permeation events and a single-channel conductance matching experiments. Additional electrostatic and kinetic modeling indicated that TMEM16As transition from outward-rectification to Ohmic conductance with increasing Ca2+ results from a weak voltage dependence of Ca2+ binding, which act cooperatively to open the pore. HIGHLIGHTSO_LIRemoval of the inhibitor 1PBC from TMEM16A induces spontaneous transitions to ion conductive and non-conductive states via bending in TM4 at two different locations. C_LIO_LISimulations suggest that the open state is stabilized by a small hydrophobic network between TM3 and 4 and disrupting this network biases channel closure in electrophysiological recordings. C_LIO_LIA kinetic model of conduction based on energetics from the all-atom MD simulations coupled to continuum calculations gives a linear current-voltage curve consistent with the fully open conformation and removal of 1 Ca2+ switches to an outwardly rectifying state via electrostatic influence on the Cl- energy profile. However, the rectification is too weak to match experiment, but a cooperative model of Ca2+ binding with weak voltage-dependence does match experiment. C_LI

Kinesin-1 trans-synaptically regulates synaptic localization of SARM1 for asymmetric neuron diversification

Khalid, A. — 2026-01-06

The Caenorhabditis elegans AWC olfactory neuron pair differentiates stochastically into two distinct subtypes, default AWCOFF and induced AWCON. A calcium signaling complex assembled by the TIR-1/SARM1 adaptor protein is transported from the AWC cell body to the axons, where it cell autonomously specifies the AWCOFF subtype through lateral signaling. UNC-104, the C. elegans homolog of the kinesin-3 motor protein KIF1A, acts non-cell autonomously in AWCON to control the synaptic localization of the TIR-1 signaling complex in promoting AWCOFF. Here, we identify a non-cell-autonomous role of unc-116/kinesin-1, similar to that of unc-104/kinesin-3, in promoting AWCOFF. unc-116 mutants, similar to unc-104 mutants, enhance the 2AWCON phenotype of a hypomorphic tir-1 mutant. Overexpression of unc-116 in AWC causes a 2AWCOFF phenotype, the same as the tir-1 overexpression phenotype. Like UNC-104, UNC-116 plays a non-cell-autonomous role in the AWCON cell to promote AWCOFF cell subtype by regulating the dynamic trafficking of TIR-1 along the AWC axon. UNC-116 is strikingly colocalized with UNC-104, while both are generally adjacent to TIR-1. Taken together, these results suggest a model in which UNC-116/kinesin-1 and UNC-104/kinesin-3 may work cooperatively to transport some unknown presynaptic factor(s) in the future AWCON cell that trans-synaptically regulates the dynamic trafficking of the TIR-1/SARM1 signaling complex to postsynaptic regions of the AWC axons in promoting the AWCOFF subtype.

A teichoic acid-like wall modification associated with immune suppression is socially regulated in Streptococcus pyogenes

Anderson, C. — 2026-01-08

Streptococcus pyogenes (Group A Streptococcus, GAS) is a human-restricted pathogen with a range of clinical manifestations and worldwide prevalence. The GAS Rgg2/Rgg3 quorum sensing (QS) system, a cell-to-cell communication network, modifies the cell surface resulting in increased lysozyme resistance, biofilm formation, and expression of the qim operon that is responsible for modulation of innate immune responses in macrophages. The operon encodes 10 genes with predicted homology to enzymes involved in bacterial cell surface-associated carbohydrate and teichoic acid biosynthesis pathways. Comparing extracts of GAS cell wall polysaccharides between wildtype and operon mutants determined that the QS-induced genes modify the S. pyogenes cell surface by adding a wall teichoic acid-like moiety of N-acetylglucosamine-linked ribitol (GlcNAc-Rbo). A fluorescently labeled phage receptor-binding protein, RBP-13-GFP, that recognizes GlcNAc-decorated ribitol phosphate repeats, bound to the GAS surface only when qim expression was induced. Deletion of the qim operon eliminated RBP-13-GFP binding, diminished bacterial colonization, and significantly attenuated GAS pathogenesis in a murine skin infection model. These findings indicate that GAS has evolved a strategy to evade innate immune response by presenting a previously unknown carbohydrate moiety upon quorum sensing. IMPORTANCEStreptococcus pyogenes is a major human pathogen, responsible for diverse clinical manifestations of both superficial and invasive infections and can lead to post-infection sequelae like rheumatic heart disease whose prevalence on a global scale rivals the most serious pathogens. Invasive S. pyogenes infections are currently on the rise worldwide, notably correlating with increasing pediatric cases of scarlet fever and enhancing the concern for long term complications. There is much that remains unknown about S. pyogenes virulence and pathogenicity, and studies focused on understanding basic systems regulating virulence factors could lead to better therapeutics and translational research. We show here one such example, where a bacterial communication system regulating a virulence mechanism relevant to in vivo infection confers the ability to alter the host innate immune response. We find that modifications to the cell wall arise when this virulence system is activated that has a direct role in host-pathogen interactions. Further research into this system could provide a mechanism for disruption and serve to treat S. pyogenes infection.

Ionic Regulation of Mechanosurveillance and Metastasis via the MRTFA/KCNMB1 Axis

Gajda, A. M. — 2026-01-14

Cellular stiffness profoundly impacts cancer metastasis at multiple levels, but mechanisms that regulate cancer cells stiffness remain poorly understood. Here, we identified potassium efflux and KCNMB1, an auxiliary subunit of the large conductance potassium efflux (BK) channels, as regulators of cellular stiffness downstream of myocardin related transcription factor A (MRTFA). In primary pericytes, KCNMB1 knockdown increased cellular stiffness, which is consistent with the role of potassium efflux in promoting relaxation during excitation-contraction coupling. In a striking contrast, however, KCNMB1 knockdown decreased cellular stiffness in cancer cells. Softer cancer cells were resistant to NK cell mediated cytotoxicity and the low KCNMB1 expression was associated with worse survival in breast cancer patients. Importantly, pharmacological activation of BK channels reduced metastatic burden in mice and improved lysis of cancer cells by cytotoxic T-lymphocytes. These results highlight the unique ionic regulation of stiffness in cancer cells and point to BK channel agonism as a new therapeutic approach in cancer.

Constructing the ensemble of representative structures for a protein via neural-surrogate-guided MSA recombination

Zhou, H. — 2026-01-15

AO_SCPLOWBSTRACTC_SCPLOWStructural dynamics is essential for the functional and mechanistic illustration of proteins. Previous research attempted to generate diversified protein structures by utilizing the multiple sequence alignment (MSA), but failed to provide physically relevant representative conformations without state annotations. In this work, we propose a framework named ProCEDiS to generate a compact ensemble of representative conformations for the target protein without prior knowledge. Adopting a neural surrogate to assist the exploration of MSA recombination and integrating with AlphaFold2 to model structures, this method can automatically find high-quality, mutually dissimilar conformations for the target sequence. Parallel short-timescale molecular dynamics (MD) simulations on these structure seeds enable quick while crude free energy estimation, from which physically plausible representative states could be identified. In the benchmark on four protein systems, the ProCEDiS + MD pipeline is capable of providing valuable structural dynamics information within acceptable running time.

RAGulate: Retrieval-Augmented Generation for Post-hoc Literature-Grounded Regulatory Assessment

Zandigohar, M. — 2026-01-21

MotivationPrioritization of transcription factor (TF)-target relationships predicted by computational models for experimental validation often requires biologists to manually inspect heterogeneous and context-dependent evidence scattered across the biomedical literature. Large Language Models (LLMs) offer a promising solution to streamline this task. However, their reliance on general-purpose knowledge may lead to hallucinations and inaccurate interpretations. ResultsWe present RAGulate, a retrieval-augmented generation (RAG) framework for literature-grounded assessment of transcriptional regulation. RAGulate leverages CollecTRI, an external regulatory knowledge base, and integrates alias-aware query expansion, sparse and dense retrieval, maximum-marginal-relevance re-ranking, and LLM-based classification of predictions within a modular pipeline. Using a balanced TF-target-context benchmark from the same resource, we evaluate retrieval, classification, and evidence faithfulness. While CollecTRI provides TF-target links with supporting PubMed Identifiers (PMIDs), RAGulate infers the context of each interaction from the retrieved literature. Results show that alias normalization markedly improves retrieval recall, while hybrid retrieval, which merges lexical and embedding-based candidates, achieves the highest evidence recovery across all cut-offs. Conditioning LLMs on retrieved documents consistently improves AUROC and AUPR for classifying whether a TF-target interaction is supported in the specified context compared with direct prompting. RAGulate reduces hallucinations and improves PMID-level citation correctness, producing explanations that faithfully reflect the supporting literature. RAGulate represents a knowledge-based AI tool that partners with biologists to accelerate the process of TF-target prioritization for experimental validation and foster hypothesis generation. Availability and implementationThe software and tutorials are available at github.com/YDaiLab/RAGulate.

Super-resolved, three-dimensional spatial transcriptomics reveals cell-type and brain-region-specific modulation of key epitranscriptomic switches following adolescent alcohol exposure

Tandukar, J. — 2026-01-23

Epitranscriptomic mechanisms dynamically regulate neuronal function through gene expression, but their precise roles in neuropsychiatric and neurological disorders remain to be fully elucidated. A major obstacle to advancing such studies is the absence of a methodology for precise, cell-type and brain-region-specific quantification of critical epitranscriptomic regulators under these complex brain conditions. To overcome this challenge, we developed a super-resolved, three-dimensional spatial transcriptomics method to quantify key epitranscriptomic switches in intact brains. Using this method, we quantified the expression of Mettl3, an N6-methyladenosine (m6A) methyltransferase enzyme recently shown to be upregulated in the amygdala after adolescent intermittent ethanol (AIE) exposure in rats. We observed a significant increase in cytoplasmic Mettl3 mRNA in neurons, but not in astrocytes or microglia, within the adult central amygdala and the CA1 and dentate gyrus of hippocampus following AIE. In contrast, no significant changes were observed across neurons, astrocytes, or microglia within the basolateral amygdala or the hippocampal CA3. Additionally, we found both the cytoplasmic density and subcellular localization of Mettl3 mRNA were dependent on the specific cell types and brain subregions examined. These results suggest that AIE increases Mettl3 expression in a highly cell-type-specific and spatially heterogeneous manner, underscoring the necessity of high-resolution spatial transcriptomics methods for studying transcriptomic and epitranscriptomic regulations under neurological conditions. Significance StatementEpitranscriptomics plays a crucial role in neuronal functions by influencing the splicing, stability, and translation of genes. However, the exact role of epitranscriptomic mechanisms, such as m6A RNA methylation, in brain disorders remains unclear, particularly in a cell-type and circuitry-specific manner. Here we developed a super-resolved, three-dimensional spatial transcriptomics method and applied it to a model of alcohol exposure. We found differential cell-type- and brain-region-specific modulation of Mettl3, a key m6A enzymatic switch, across major brain regions following adolescent intermittent ethanol exposure in adulthood. Our findings, coupled with our pipeline, are expected to address existing methodological limitations and knowledge gaps, thereby accelerating brain transcriptomic and epitranscriptomic studies under various psychiatric and neurological conditions.

A high fermentable fiber Western diet reduces indole levels

Priyadarshini, M. — 2026-01-30

Changes in gut microbiota composition due to diet impact health. Fiber-rich diets promote beneficial microbiota and reduce the risk of metabolic diseases, while low-fiber, calorie-dense diets are linked to dysbiosis and increased disease risk. This study examines the effects of a Western diet (WD) and explores dietary fiber supplements as potential modifiers of those effects. 10-week-old C57Bl/6J male mice were fed control (low-fat) or WD (high-fat, high-sucrose) containing 0% fermentable fiber (FF) or WD supplemented with 20% FF (fructooligosaccharides, FOS; guar gum, GG, or pectin, Pec). After 19 weeks, analysis of the cecal metagenome using whole-genome shotgun sequencing, metabolome by untargeted and targeted LC-MS/MS, and tissue RNA and protein expression by RT-PCR and immunoblotting was undertaken. WD-FF reduced metabolic derangements from WD while also improving GM diversity and altering cecal metabolites, particularly tryptophan metabolism. A profound increase in cecal indole levels (targeted metabolomics) was noted in WD vs WD-FF groups. As the primary indole-oxidizing enzyme, CYP2E1 generates indoxyl sulfate, which contributes to oxidative stress and a leaky gut. Mice on WD displayed higher expression of Cyp2e1 mRNA in the gut. In the liver, the levels of both CYP2E1 protein and mRNA were higher in the WD group compared to the WD-FOS group, with protein levels also higher than in the WD-Pec group and mRNA levels higher than in the WD-GG group. mRNA expression of markers of oxidative stress, inflammation, and leaky barrier was significantly higher in the liver and intestine of the WD vs the WD-FF groups. FFs reduced high plasma indoxyl sulfate levels (except in WD-GG), and boosted short-chain fatty acids and indole acetic acid. Our data suggest that WD disrupts GM tryptophan metabolism, possibly by altering the balance between indole-producing and utilizing gut bacteria. Dietary fiber supplementation exerts protective effects, in part, by mitigating this imbalance.

Pseudotime graph diffusion for post hoc visualization of inferred single-cell trajectories

Lukas, B. E. — 2026-01-30

Visual representations are widely used to interpret trajectories in single-cell data; however, they do not always faithfully capture inferred trajectory structure. As a result, interpretation of cellular dynamics and downstream analyses may be compromised. Here, we present Pseudotime Graph Diffusion (PGD), a lightweight and interpretable post hoc framework for smoothing cell-level features along pseudotime. PGD operates by performing random-walk diffusion on a pseudotime graph, propagating information along inferred trajectory paths to enhance continuity and structure. We demonstrate that PGD-smoothed embeddings improve visualization of increasingly complex inferred trajectories of monocytes and macrophages during wound healing. We further show that PGD extends naturally to trajectory-aware gene expression smoothing. By improving agreement between visual representations and inferred trajectories, PGD enables more faithful interpretation of dynamic cellular processes.

Arginine Kinase 1 regulates energy homeostasis in Drosophila muscle development

Zappia, M. P. — 2026-02-02

In Drosophila, Arginine kinase 1 (Argk1) is involved in maintaining ATP homeostasis during bursts of activity in tissues with high and variable rates of energy turnover such as muscle. However, its role beyond stress conditions is less understood. Here, we show that Argk1 maintains energy homeostasis during flight muscle development and is required for animal viability and proper muscle function. The knockdown of Argk1 causes defects in both early and late stages of myogenesis. In the proliferating myoblasts associated with the wing disc, Argk1 depletion results in a reduction in cell size without changes in cell cycle progression. Single cell RNA-sequencing revealed that the overall composition of differentiating and undifferentiating myoblasts is not altered. Nonetheless, Argk1 knockdown causes broad alterations in the expression of genes involved in various metabolic pathways. This correlates with low levels in both ATP content and NAD+/NADH ratio. Later in muscle development, Argk1-depleted muscles completely lack spontaneous muscle contractions that are essential in myofibrillogenesis. Accordingly, Argk1 knockdown results in severe defects in sarcomere structure, while the mitochondrial network is highly fragmented. Furthermore, muscle growth is severely reduced. Thus, our data reveal an essential role for Argk1 in maintaining energy homeostasis throughout muscle development, which is required to meet the demand to support myofibrillogenesis, muscle growth and proper muscle function.

A comparative analysis of co-contraction indices using synthetic EMG data: Implications for selection and interpretation

Carey, H. D. — 2026-02-05

Co-contraction--the simultaneous activation of opposing muscles--influences movement efficiency, joint stability, and motor learning. While consensus exists for EMG acquisition and processing, comparable guidance for quantifying co-contraction is lacking. This study evaluated the behavior and interrelationships of six commonly used co-contraction indices (CCIs) to develop practical recommendations for their selection, calculation, and interpretation. Synthetic EMG-like signals were generated and used to evaluate CCI behavior across a range of conditions that would be difficult to achieve experimentally. Based on their formulas and observed behavior, CCIs were sorted into three categories: shape-based, amplitude-driven, and temporal indices. Shape-based CCIs increase when two EMG signals have similar shapes, regardless of amplitudes. Amplitude-driven CCIs increase when activation is high in both muscles. Temporal CCIs increase as the duration of EMG overlap increases, regardless of signal shape or amplitude. Correlation analyses showed stronger associations within-category than between-category, supporting the proposed classification scheme. CCI behavior yielded three principal findings, each paired with a practical recommendation. First, EMG amplitude normalization techniques altered co-contraction estimates, and the effect varied by index. Researchers should therefore test whether their conclusions hold across normalization methods. Second, because CCIs differ in scale and theoretical maxima, their values are not directly comparable across indices. Comparisons should instead focus on relative trends interpreted within each indexs bounds. Third, each CCI category was sensitive to different EMG features (e.g., amplitude versus shape). The choice of CCI should therefore align with hypothesized differences in EMG signals - use shape-based CCIs when waveform similarity is of interest, and amplitude-driven CCIs when differences in activation magnitude are expected. These results provide initial guidance for selecting, calculating, and interpretating CCIs, and they establish a framework for testing the robustness of these theoretical findings using experimental EMG from diverse tasks, muscle pairs, and populations.

Microvascular network organization and hemodynamic perfusion protect the brain against hypoxia

Ventimiglia, T. A. — 2026-02-06

Mechanistic simulations of blood flow and oxygen exchange showed regions of cortical tissue tolerating substantial increase in local oxygen consumption (CMRO2) before reaching hypoxia (pO2<10 mmHg). The observed robustness in O2 supply was attributed to overcapacity in convective oxygen transport in the pial arterial network combined with a surplus in the number of capillary flow paths. Microcirculatory flux analysis suggests that network induced hemodynamic flow patterns impart intrinsic reserve to protect the brain against perfusion variances or metabolic demand surges during activation. Furthermore, oxygen transport in cortical tissue is characterized by two regimes: in the transport zone -- centered on penetrating arteriole trees composed of a single penetrating vessel connected to the post-arteriole capillary transition zone -- strong diffusion supports high oxygen tension with only modest contribution from capillaries. This regime transitions into the terminal/reactive zone where oxygenation is sensitive to capillary density and perfusion. Quasi-dynamic simulations also enabled reconstruction of the BOLD signal underlying functional imaging. Simulations at single micron resolution further show that age-related reductions in arterial saturation and systemic hematocrit were sufficient to induce hypoxic tissue pockets in the terminal zone at nominal perfusion (CBF) and metabolic activity (CMRO2), and neutrophil adhesion induced capillary flow stalling further exacerbates hypoxia.

Th17 effector cytokines induce shared and distinct microglial and endothelial cell responses in post-streptococcal encephalitis

Wayne, C. R. — 2026-02-07

Group A Streptococcus (GAS) infections can lead to neuropsychiatric sequelae in children, yet the mechanisms driving post-infectious brain pathology remain poorly defined. In a mouse disease model, Th17 lymphocytes induce microglial activation, blood-brain barrier (BBB) dysfunction, and neural circuit impairment; however, the transcriptional programs underlying these effects, and the specific Th17-derived cytokines involved are unclear. Using mouse genetics, single-cell RNA sequencing, and spatial transcriptomics, we show that GAS infections induce inflammatory gene programs in microglia and brain endothelial cells (BECs), accompanied by downregulation of BBB-associated transcripts in BECs. Spatial transcriptomic analyses reveal that GAS-responsive microglia are enriched near infiltrating T cells. Several chemokines upregulated in microglia following GAS infection in mice are elevated in sera from affected patients. Conditional ablation of GM-CSF in CD4+ T cells partially attenuates microglial chemokine gene expression, but does not restore BBB integrity. Neutralization of IL-17A partially rescues BBB transcriptional changes in BECs and reduces microglial chemokine expression; however, compensatory peripheral immune responses associated with persistent infection exacerbate BBB disruption. In contrast, microglia/macrophage-specific deletion of IL-17 receptor A partially rescues BBB deficits following GAS infection. Together, these findings identify IL-17A-IL-17RA signaling in microglia as a critical driver of BBB dysfunction after GAS infections.

Innate Immune Function of Neutrophil Cytoplasts Generated Post-Vital NETosis

Prasad, N. R. — 2026-02-09

The migration of polymorphonuclear neutrophils (PMN) into the site of infection such as lungs during pneumonia is a canonical feature of innate immunity. Formation of neutrophil-derived extracellular traps (NETs), web-like strands of varying lengths comprising DNA, histones, elastase, and myeloperoxidase, is an important determinant of PMN-mediated innate immunity. NETs form in microvessels, entrap bacteria and effete matter, and dampen PMN-mediated inflammatory injury at specific sites. However, studies have largely focused on NET release secondary to lytic NETosis and lysis of PMN. Far less is known about vital NETosis occurring in the absence of PMN rupture. As vital NETosis is characterized by generation of anuclear PMN termed cytoplasts (PMNcyto), we addressed the function of PMNcyto as a critical determinant of PMN-mediated innate immunity. Studies were made in mice challenged with live Pseudomonas aeruginosa (PA) i.t. to induce fulminant pneumonia characterized by tissue injury in which we determined the role of generated PMNcyto population. Using Tomato Red (tDTomato) transgenic mice to mark PMN, we observed PA pneumonia induced PMN transmigration leading to PMNcyto generation in the airspace. In contrast, PMNcyto transmigration, was minimal. PMNcyto accumulating in lung tissue actively phagocytosed and killed PA. Instillation of ex vivo generated PMNcyto also prevented PA-induced inflammatory lung injury and reduced mortality as compared to control mice. We demonstrated that the salutary effects of PMNcyto required functional microchondria. Proteomic analysis revealed that PMNcyto retained bactericidal and ROS generating pathways, consistent with an intact plasma membrane. Genetic deletion of peptidyl arginine deaminase 4 (PAD4), which mediates histone citrullination and promotes NETosis, facilitates PMNcyto generation and thereby abrogated pneumonia-induced mortality. Thus, we have identified the crucial host defense function of PMNcyto generated post-vital NETosis, suggesting that PMNcyto hold promise as cell based anti-bacterial therapy in pneumonia.

4,5-dihydroxyhexanoic acid is a robust circulating and urine marker of mitochondrial disease and its severity

Skinner, O. S. — 2026-02-12

Management of patients with mitochondrial respiratory chain diseases is challenging, in part because of our incomplete understanding of pathogenesis and a lack of biomarkers. Unknown metabolites account for >90% of detected features in modern metabolomics experiments and hold immense untapped promise for new basic and biomedical research. We recently used mass spectrometry-based metabolomics to identify and validate 19 circulating blood-based biomarkers for patients with the mitochondrial DNA (mtDNA) m.3243A>G pathogenic variant, which is the most frequent cause of the mitochondrial disorder MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). However, the most significantly changing biomarker corresponded to an "unknown" metabolite. Here, we combine cheminformatics with analytical chemistry and identify that feature as 4,5-dihydroxyhexanoic acid (4,5-DHHA), a metabolite previously associated with inherited defects of gamma-aminobutyric acid (GABA) catabolism, but with no prior links to mitochondrial respiratory chain disorders. We validate this finding in an independent MELAS cohort and further show that 4,5-DHHA levels correlate with disease severity and are elevated in patients with other forms of mitochondrial disease and sepsis. Furthermore, brain 4,5-DHHA levels were elevated in two genetic mouse models of mitochondrial disease. In vitro and tissue culture experiments indicate that 4,5-DHHA is generated when the GABA catabolite succinic semialdehyde reacts with an intermediate of the pyruvate dehydrogenase reaction and is sensitive to mitochondrial complex I function. Our work identifies 4,5-DHHA as a robust plasma and urine marker of mitochondrial dysfunction in humans and reveals new connections between the respiratory chain and GABA metabolism. Significance StatementInborn errors of the mitochondrial respiratory chain cause severe, progressive diseases, yet effective treatments and biomarkers remain limited. Modern metabolomics detects thousands of molecules in biofluids, but the vast majority are unidentified. In this study, we investigate the most significantly altered blood metabolite in patients with the most common mitochondrial disease - MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) - and identify it as an 4,5-dihydroxyhexanoic acid (4,5-DHHA). We show that 4,5-DHHA is reproducibly elevated and correlates with severity. Levels are increased across multiple mitochondrial disorders as well as in sepsis and rise when respiratory chain function is impaired. These findings establish 4,5-DHHA as a promising biomarker of mitochondrial dysfunction and reveal a link to dysregulated GABA metabolism.

AAV-only targeting of ventral tegmental area dopamine neurons for optical self-stimulation studies in mice

Marron Fernandez de Velasco, E. — 2026-02-12

Studies employing optogenetic approaches in rodent models have highlighted the important contribution of ventral tegmental area (VTA) dopamine (DA) neurons to reward, learning, and motivation. Selective manipulation of VTA DA neurons is generally achieved in these studies using transgenic mouse or rat lines that express Cre recombinase under the control of a promoter active in DA neurons, combined with intra-VTA infusion of adeno-associated virus (AAV) vectors harboring Cre recombinase-dependent expression cassettes. Reliance on transgenic Cre driver lines is expensive and decreases study efficiency, and available driver lines have unique limitations. Here, we report the development of an AAV-only approach that permits genetic access to VTA DA neurons and can support optogenetic self-stimulation in mice. We used a 2.5 kb fragment of the mouse tyrosine hydroxylase promoter (mTH) to drive Cre expression in VTA DA neurons. Intra-VTA co-infusion of AAV8-mTH-Cre with an AAV vector harboring a Cre-dependent yellow fluorescent protein expression cassette yielded high efficiency (82%) and high fidelity (73%) targeting of tyrosine hydroxylase-positive VTA neurons in C57BL/6J mice. Co-infusion of AAV8-mTH-Cre with a vector harboring a Cre-dependent channelrhodopsin (ChR2) expression cassette permitted optical regulation of VTA neurons with electrophysiological features consistent with VTA DA neurons. Moreover, C57BL/6J mice expressing ChR2 in VTA DA neurons rapidly acquired optical self-stimulation behavior. Thus, this AAV-only approach should facilitate investigation of VTA DA neuron contributions to reward-related behaviors and permit comparative assessments in reward circuit function in inbred and mutant mouse strains.

BLIMPS: a technique for tandem biosensor imaging across multiple populations of presynaptic terminals, using lattice light sheet microscopy.

Potcoava, M. — 2026-02-15

Within neuronal circuits, ordered neurotransmission is contingent upon balance between excitatory glutamatergic and inhibitory GABAergic signaling. To study circuit-level processes, the paradigm of 4D cellular physiology has been developed, where, single cells and subcellular structures are studied as individual units in three-dimensional space over a continuous interval rather than as a single moment in time, or as a population-level average. Neurons are excitable cells expressing voltage-gated Ca2+ channels and Ca2+ fluxes subsequent to action potential firing are widely used as markers of neuronal activity. While the imaging of Ca2+ dynamics at the soma is often performed, the imaging of Ca2+ fluxes at presynaptic terminals has often proven to be an experimental challenge: existing imaging modalities suffer from inadequate acquisition speeds, insufficient penetration depths, insufficient spatial resolution to identify axonal structures, or spectral crosstalk issues. To visualize presynaptic Ca2+ dynamics in both excitatory and inhibitory neurons, here we combine advanced lattice light-sheet microscopy with viral delivery of two genetically encoded calcium indicators (GECIs)- jRGECO1a and jGCaMP8f, to perform sequential imaging of Ca2+ dynamics within acute ex vivo slice preparations. Our methodology, Biosensor Lattice light-sheet Imaging of Multidimensional Presynaptic Structure (BLIMPS), includes acute brain slice preparation, mounting on a temperature-controlled flow chamber within a LLSM, and imaging of electrically evoked Ca2+ signals, with high adaptability to a range of genetic and pharmacological disease models. Our technique offers high spectral separation between evoked signals from each of the two GECIs and fast acquisition speeds of 0.1-0.3 KHz. Included within the BLIMPS technique is a robust, open-source data analysis pipeline to track highly responsive neuronal structures such as presynaptic terminals and quantify both the amplitudes and decay rates of evoked fluxes.

Vitamin D deficiency alters prostate epithelial differentiation and increases prostate cancer aggressiveness in ex vivo and in vivo models

Duraki, A. — 2026-02-18

Here, we examined the consequences of biologically relevant vitamin D deficiency, a known risk factor for aggressive prostate cancer, using ex vivo and in vivo models. Phenotypic and single-cell RNA sequencing of mouse prostate organoids showed that vitamin D deficiency stunted luminal cell differentiation more than androgen deficiency, which is a known driver of prostate development. Mice fed a vitamin D-deficient diet showed significantly altered expression of androgen-responsive genes in their prostate luminal cells, as determined by single-cell RNA sequencing. MDA-PCa-2b human prostate cancer cells, when maintained for 6 months in 1,25-dihydroxyvitamin D, lost the ability to form xenografts, despite normal proliferation in vitro. RNA sequencing showed that these cells also had disruptions in androgen signaling and multiple cancer-related pathways. This study offers new insights and validation of vitamin Ds role in both benign and malignant prostate biology, underscoring its essential hormonal functions and supporting strategies for vitamin D supplementation to reduce prostate cancer risk in vulnerable populations. STATEMENT OF SIGNIFICANCEVitamin D is an essential hormone, however, the non-calcemic consequences of vitamin D deficiency remain poorly defined, despite its high prevalence in the population. This study demonstrates significant biological consequences of vitamin D deficiency on prostate cells at biologically relevant levels in multiple systems.

Regulatory network architecture constrains inflammatory responses in tissue-resident alveolar macrophages

Kruszelnicki, S. — 2026-02-18

Macrophages across different tissues exhibit remarkable functional diversity while maintaining core innate immune-cell programming. These complex programs are governed by gene regulatory networks, in which precise transcription factor activity tunes the expression of functionally relevant gene modules. Although the contributions of individual transcription factors have been well characterized, the higher-order regulatory interactions that coordinate tissue-resident macrophage identity and inflammatory response regulation remain poorly understood. Here, we integrate single-cell RNA-seq data with ATAC-seq profiling and deep-learning-based chromatin accessibility modeling to infer gene regulatory network architectures in tissue-resident versus recruited monocyte-derived alveolar macrophages under inflammatory stress. Our results suggest that inflammatory responses are more restrained in tissue-resident alveolar macrophages compared with recruited macrophages due to a stabilizing regulatory network architecture involving PU.1 and CEBP/{beta}. This work advances our understanding of functional plasticity in tissue-resident macrophages and their role in host defense.

Structural modification of oxazolidinone antibiotics alters nascent peptide stalling preference and peptide trajectory through the ribosome

Kleinman, J. I. — 2026-02-18

The oxazolidinone antibiotic linezolid binds to the peptidyl transferase center of the ribosome, where it inhibits a subset of peptide bond formation events. This context-specificity of translation inhibition is dictated by the nature of the amino acid at the penultimate position of the nascent peptide. It remains unknown whether this is a general feature of oxazolidinones and whether it can be modulated by their structural alterations. Here, we show that the oxazolidinone tedizolid also inhibits translation in a context-specific manner, but with dramatically altered selectivity, favoring Ile, His, and Gln as the penultimate residues. Delpazolid, which shares the C5 hydroxymethyl moiety with tedizolid, shows a similar preference. Structural analysis of the ribosome with tedizolid and a stalled nascent peptide showed a compacted, helical conformation of the nascent chain induced by the drug. Our findings reveal that stalling preferences of oxazolidinones can be modulated by structural modifications within this antibiotic class.

Cryo-electron tomography reveals paracellular claudin-15 pores at the tight junction

Demchenko, E. — 2026-02-20

Tight junctions (TJs) are composed of anastomosing strands between epithelial cells. Members of the claudin family of proteins reside within TJ strands and either seal the paracellular space or assemble into charge and size-selective pathways. Functional studies suggest that claudin-mediated conductance pathways resemble traditional ion channels. However, such postulated pores have not been directly visualized. Using a model claudin deficient epithelium where exogenously introduced EGFP-CLDN15 is the only claudin family member expressed, our investigation sheds light on the arrangement and structure of the postulated claudin pores. Following correlative light and electron microscopical identification of TJs and cryo-electron tomography, we identified series of linearly distributed electron lucent features that locate between two closely apposed plasma membranes of adjacent cells. At these sites, the median spacing between adjacent features is 2.25 nm (IQR = 1.83), with a median 1.66 nm (IQR = 0.92) diameter. In contrast, such features were not observed in claudin deficient model epithelium with exogenous mCherry-ZO-1 expression. These findings agree with the postulated and extensively modeled claudin pores formed within the simple columnar epithelium. This provides the first direct evidence of paracellular pore organization and paves way for future biophysical investigation. SIGNIFICANCEBy combining correlative fluorescence imaging, FIB milling, and cryo-ET within an epithelial system restricted to a single claudin isoform, we were able to visualize repetitive, low-density pore features within CLDN15-containing tight junctions (TJs), structures not previously resolved in intact epithelia. These features were absent in claudin-negative controls and displayed placement and geometry consistent with CLDN15 X-ray crystallography and molecular dynamics models. Quantitative measurements of pore diameter, paracellular gap width, and pore spacing further support their assignment as CLDN15 pores. These findings establish a structurally validated platform for defining claudin pore ultrastructure and provide a foundation for future efforts to compare pore-forming and barrier-forming claudins, understand disease-associated junction remodeling, and guide therapeutic modulation of epithelial barrier function.

Explaining the unexplained admixture mapping signals via rare variants: the HCHS/SOL

Chen, X. — 2026-02-23

In admixed populations, formed by a mixing of two or more previously isolated populations, genomic segments can be traced to their ancestral populations ("ancestries"). Admixture mapping (AM) associates local ancestry with outcomes in admixed populations, detecting signals when causal variants differ in frequency or effect across ancestral populations. Prior work showed that adjusting for nearby GWAS-identified common variants does not fully explain some AM signals. Here, we assessed two approaches to explain the previously unexplained AM signal: (1) including sets of rare variants; (2) increasing the genomic region considered when searching for common variants. We studied these hypotheses comprehensively using a whole-genome sequencing dataset coupled with metabolomics from the Hispanic Community Health Study/Study of Latinos. We detected multiple sets of rare variants with replicated association with metabolite levels. Yet these rare variants appear to explain only a small fraction of the AM signal, while inclusion of common variants from a larger genomic region appears to explain the majority of the AM signals.

Impact of Aging, Sex, and Species on the mRNA Expression of Matrix Metalloproteinases Following Ischemic Stroke

Challa, S. R. — 2026-02-26

Matrix metalloproteinase (MMP) expression and function are highly context dependent, varying across physiological and pathological conditions. We previously documented the expression profiles of select MMPs in the ischemic brains of young male rodents. However, aging is a major risk factor for stroke in humans and is associated with vasculature alterations, increased oxidative stress, and elevated inflammation. In addition, sex differences have been reported in stroke incidence and severity. Despite this, the effects of age, sex, and species on brain MMP gene expression after cerebral ischemia/reperfusion (I/R) has not been systematically examined. Therefore, we investigated how age, sex, and species influence the mRNA expression of all known MMPs (22 total) in the brain following cerebral I/R. Moderate-to-severe neurological deficits were induced by transient middle cerebral artery occlusion (MCAO) followed by reperfusion in young and aged male and female C57BL/6 mice and in young male Sprague-Dawley rats. Brain tissue from the ipsilateral (ischemic) hemisphere was collected on post-MCAO day 1, and MMP mRNA levels were quantified by real-time PCR and expressed as fold change relative to the sham control group. Across species, MMP-3, MMP-8, MMP-12, MMP-13, MMP-19, MMP-20, and MMP-27 were upregulated in both rats and mice. Species-specific increases were also observed: MMP-1, MMP-7, MMP-9, MMP-14, MMP-21, and MMP-25 were upregulated only in rats, whereas MMP-10 was upregulated only in mice. The most strongly upregulated MMPs were MMP-12 in rats and MMP-3, MMP-10, and MMP-12 in mice. By contrast, MMP-15 and MMP-17 were downregulated in both species, whereas MMP-23 and MMP-24 were downregulated only in rats and mice, respectively. Within mice, MMP-3, MMP-10, MMP-12, MMP-19, MMP-20, and MMP-21 increased in both sexes and age groups, except for MMP-19 in aged males and MMP-21 in young males. MMP-14 increased only in females (young and aged), whereas MMP-27 increased only in males (young and aged). Notably, MMP-3, MMP-10, and MMP-12 were the three most highly upregulated MMPs in both male and female mice regardless of age. Overall MMP mRNA expression levels were higher in aged male mice and lower in aged female mice relative to sex-matched young mice. Among all MMPs examined, MMP-12 showed the most marked upregulation across species and, within mice, across age groups and sexes. Collectively, these findings demonstrate that brain MMP gene expression after cerebral I/R is modulated by age, sex, and species, underscoring the importance of incorporating these biological variables when targeting MMPs individually or in combination in preclinical rodent stroke models.

Counteraction of HMGB1 at ss-dsDNA junctions maintains liquidity of protamine-DNA co-condensates

Ahlawat, V. — 2026-02-26

In the sperm nucleus, protamine replaces histones to mediate extreme DNA compaction. The histone-to-protamine transition involves the occurrence of double-strand breaks, and is facilitated by transition proteins including those containing high-mobility-group (HMG) boxes. Here we used optical tweezers and microscopy to study the actions of HMGB1 and protamine on DNA. Confocal scans of GFP-HMGB1 on overstretched {lambda}-DNA show 2-3 foci that spread on the DNA upon retraction. Spreading of foci coincides with reannealing of ssDNA tracks, confirming their localization at ss-dsDNA junctions. Whereas the force-extension curves of protamine-bound {lambda}-DNA show tangles that withstand forces > 60 pN, premixing protamine with HMGB1 produces only bends and bridges ([~] 20 pN). The counteraction of HMGB1 involves its acidic C-terminal tail, as HMGB1-{Delta}C fails to prevent tangle formation. In line with these single-molecule results, brightfield and confocal imaging shows that HMGB1 converts protamine-dsDNA aggregates into liquid droplets whereas HMGB1-{Delta}C fails to do so. Together, these observations support our hypothesis that chromatin-associated proteins like HMGB1 help maintain early protamine-mediated DNA condensates in a liquid state, enabling the recruitment of the repair machinery to restore the duplex structure.

A Novel Monocyte-derived Antigen Presenting Cell-T regulatory Cell Axis Contributes to Skin Wound healing and is Impaired in Diabetic Mice

Pang, J. — 2026-03-06

Despite a vast literature on the role of macrophages in wound healing, the role of dermal monocyte (Mo)-derived antigen presenting cells (APC) has received scant attention. Using scRNAseq and flow cytometry, we identify a population of APC that is prominent in wounds of non-diabetic mice but is reduced in wounds of diabetic mice. Using adoptive transfer experiments and Ccr2 knockout mice, we demonstrate that wound APC are derived primarily from Mo and that the diabetic wound environment inhibits differentiation of Mo into APC. We also show that Mo-specific Irf4 knockout mice exhibit reduced differentiation of Mo into APC, decreased levels of IL27 and numbers of activated Treg cells in wounds. and impaired wound healing. Importantly, adoptive transfer of bone marrow Mo that express Irf4 into wounds of Mo-specific Irf4 knockout mice rescued levels of wound APC and activated Treg, as well as wound healing. Local administration of recombinant IL27 into wounds of these mice also rescued levels of activated Treg in wounds, along with wound healing, Together, these findings identify a novel pathway in which IRF4 induces Mo differentiation into APC in wounds, which in turn produce IL27 that activates Treg to promote healing. This pathway is impaired in wounds of diabetic mice, which provides a novel target to improve diabetic wound healing.

IFI207 promotes antiviral responses by modulating STING ubiquitination and degradation

Enya, T. — 2026-03-07

Aim2-like receptors (ALRs) play crucial roles in innate immune signaling pathways and demonstrate strong positive selection likely driven by pathogens. IFI207, an ALR found in all Mus species, enhances interaction with and stabilization of STING, contributing to the control of Murine Leukemia Virus (MLV) infection. We show here that IFI207 enhances the type 1 interferon response by inhibiting activation-induced K63-linked ubiquitination of STING, thereby preventing its recognition by hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a key component of the ESCRT complex, and its subsequent degradation in lysosomes. IFI207 promotes downstream signaling in the STING pathway in multiple cell types and moreover enhances the STING-dependent response to herpesvirus simplex 1 infection ex vivo and in vivo. We also show that IFI207 likely functions in dendritic cells to suppress MLV infection. Our study reveals that IFI207 acts as a modulator in the STING pathway, strengthening the hosts defense against viral infections and suggests that the expansion of the Alr locus in mice may have occurred in response to endemic viruses.

Hippocampal representations of temporal structure increase in scale and symmetry across development

Friend, O. W. — 2026-03-11

Learning which experiences reliably co-occur in time is fundamental to episodic memory and improves markedly across childhood and adolescence. Although children and adults both engage the hippocampus while learning predictable sequences, the nature of the neural representations supporting statistical learning across development remains unknown. Here, we directly quantified item-level neural representations before and after children, early adolescents, and adults learned predictable temporal relationships between items, providing a direct measure of learning-related representational change. We identified three developmental shifts in hippocampal representation. First, although posterior hippocampus integrated temporally adjacent sequence elements similarly across age groups, integration of non-adjacent sequence elements increased with age in anterior hippocampus, indicating developmental expansion in the temporal scale of neural integration. Second, hippocampal representations changed in their directional organization, with children showing hippocampal representations reflecting only forward associations between adjacent events, whereas adolescents and adults exhibited bidirectional integration of sequence relationships. Third, functional connectivity between anterior hippocampus and frontoparietal cortex tracked statistical transition probabilities during learning and predicted memory performance. Together, these findings show that improvements in statistical learning during development reflect reorganization of hippocampal representations and hippocampal-cortical interactions, revealing how the developing brain constructs increasingly flexible representations of predictive temporal structure. HighlightsO_LIHippocampal representations of temporal sequences reorganize across development C_LIO_LITemporal integration expands from adjacent to non-adjacent events with age C_LIO_LISequence representations shift from forward-only to bidirectional integration C_LIO_LIHippocampal-frontoparietal connectivity predicts statistical learning ability C_LI

Flow-sensitive K+ channels link flow to piezo1/PI3K/Akt1 pathway

Ahn, S. J. — 2026-03-12

BackgroundEndothelial response to flow is key to vascular function in health and disease. Our earlier studies demonstrated that endothelial Kir2.1 is essential for flow-induced Akt1/eNOS signaling and for flow-induced vasodilation (FIV) but the mechanistic integration between Kir and other flow signaling pathways remained poorly understood. MethodsWe use a combination of electrophysiological recordings in real time of flow exposure, Ca2+ imaging, pressure myography of resistance arteries, and echocardiography. ResultsWe demonstrate that Kir2.1 is essential for flow-induced PI3K phosphorylation, whereas expression of myristoylated Akt1, which bypasses PI3K-dependent membrane recruitment, restores flow-induced Akt1/eNOS phosphorylation in Kir2.1-deficient endothelium. It also restores FIV in Kir2.1-deficient mesenteric arteries. We further demonstrate that Kir2.1 is essential for flow-induced Ca{superscript 2} influx mediated by Piezo1 and TRPV4 channels, whereas Ca{superscript 2} influx induced by pharmacological activation of these channels is Kir2.1 independent. Deficiency of Piezo1 does not affect endothelial Kir2.1 channels. We also discover that flow activation of endothelial Kir2.1 requires Syndecan1, thus creating a link between glycocalyx and downstream effects. Physiologically, we find that endothelial Kir2.1 is suppressed by infusion of Angiotensin-II and by advanced aging, resulting in significant impairment of FIV. In both cases, FIV is fully restored by endothelium-specific over-expression of Kir2.1. ConclusionsOur study reveals that Kir2.1 serves as a mechanistic linker between endothelial glycocalyx to Piezo1-mediated Ca2+ influx and downstream signaling suggesting a new integrated model of endothelial mechanotransduction. A functional loss of endothelial Kir2.1 is shown to play a significant role in FIV impairment in Angiotensin-induced hypertension and aging.

Epstein-Barr virus induced epigenetic reprogramming drives cancer stem cell emergence in breast cancer

Friedenson, B. A. — 2026-03-12

Basal breast cancers display stem cell associated basal programs, but originate from luminal progenitors. This lineage paradox may be created by Epstein-Barr virus (EBV) infection. Across more than 2,000 breast cancer genomes, coordinated methyla-tion changes appeared in cis-regulatory elements governing stem cell differentiation. Methylation positions followed EBV-associated malignancies with striking accuracy independent of whether ER-status marked a luminal or basal cancer. EBV-driven epigenetic reprogramming was incompatible with tumor infiltrating lymphocytes and disrupted lineage specification before tumorigenesis. Breast cancers commonly showed coordinated viral response indicators that tracked with antigen presentation and stem cell differentiation programs. Non-malignant keratinocytes with resolved EBV infections retained some aberrantly methylated loci. Analyses of non-EBV skin carcinoma, randomized genomic sites, endogenous retroelements, DUX4, and repli-cation clocks confirmed the specificity of EBV-linked alterations. These findings posi-tion EBV as a developmental lineage hijacker that reprograms cells into premalignant stem-like states. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/710870v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@13c8d52org.highwire.dtl.DTLVardef@620426org.highwire.dtl.DTLVardef@fe9a96org.highwire.dtl.DTLVardef@156f170_HPS_FORMAT_FIGEXP M_FIG C_FIG EBV infection reprograms the methylome of breast luminal progenitors to generate or select for cancer stem-like cells. In BriefEpstein-Barr virus (EBV) rewires developmental programs that specify cell identity, creating cancer stem-like cells with malignant potential. This targeted reshaping of progenitors links viral infection to the earliest steps of breast cancer development. HighlightsO_LIEpstein-Barr virus (EBV) infection reprograms the methylome of lu-minal progenitors generating breast cancer stem-like cells. C_LIO_LIEBV-driven methylation sites overlap luminal and basal breast cancer signatures that regulate stem cell differentiation. C_LIO_LICharacteristic responses to viral infection are common in breast can-cer and they correlate with gene programs needed to differentiate progenitor cells. C_LIO_LIEven after viral clearance, EBV leaves persistent methylation scars in non-malignant cells that may increase risks for future malignancy. C_LI

A membrane insertion code for intrinsically disordered proteins

Muhammedkutty, F. K. — 2026-03-13

Membrane association of intrinsically disordered proteins (IDPs) mediates various cellular functions including membrane remodeling and signal transduction. Whereas membrane association through amphipathic helices and polybasic motifs is well understood, sequence determinants for deep membrane insertion of aromatic residues are still poorly characterized. Here, we decipher the sequence code for membrane insertion of aromatic-centered motifs. For an initial set of 10 9-residue aromatic-centered sequences, all-atom molecular dynamics simulations and the positioning of proteins in membranes (PPM) method produced very similar membrane insertion propensities. Applying PPM to a full library of 1.2 x 106 sequences with an F, W, or Y residue flanked by L, R, G, N, or E at four positions on either side, we found that aliphatic (L) and basic (R) residues favor membrane insertion, whereas acidic (E) and polar (N) residues disfavor it. Guided by these rules, we developed a mathematical model dubbed AroMIP to predict the membrane insertion propensities of aromatic-centered motifs. AroMIP achieves 91.2%, 92.0%, and 99.7% accuracies for F-, W-, and Y-centered motifs, respectively, in disordered regions of the human proteome and is available as a web server at https://zhougroup-uic.github.io/AroMIP/. The present work provides the sequence basis and a mechanistic understanding of how IDPs employ aromatic-centered motifs to drive membrane insertion, and enriches the tools for the study of IDP-membrane association. Significance StatementMembrane insertion of short motifs, along with membrane tethering of amphipathic helices and membrane binding of polybasic motifs, is a major mode of membrane association and mediates diverse functions including membrane remodeling and signal transduction. Here we used three complementary approaches to decipher the sequence code of membrane insertion, culminating in a sequence-based method, AroMIP, for predicting membrane-insertion propensities. Aromatic sidechains have the intrinsic ability to insert deeply into the acyl chain region of membranes; at flanking positions, they strongly stabilize the inserted state. Aliphatic and basic residues are medium to modest promoters of membrane insertion. AroMIP has >90% accuracy and identifies important motifs for regulating functions of intrinsically disordered proteins via membrane insertion.

An apical junction protein antagonizes mechanosensitive calcium signaling to establish stochastic choices of olfactory neuron subtypes

Xiong, R. — 2026-03-16

Mechanical forces regulate brain development and left-right body patterning. However, the role of mechanical signaling in brain lateralization remains unclear. In Caenorhabditis elegans, the left and right AWC olfactory neurons communicate via a gap junction network to stochastically differentiate into the default AWCOFF and induced AWCON subtypes. SLO BK potassium channels, SLO-1 and SLO-2, act redundantly to inhibit a calcium-regulated protein kinase pathway in the specification of the AWCON subtype. Here, we identified a role for AJM-1 (apical junction molecule 1) in promoting AWCON from an unbiased forward genetic screen for mutants that suppress the slo-1(gf) 2AWCON phenotype. AJM-1 is located at three distinct tight junctions between amphid neurons (including AWC) and sheath glia, sheath and socket glia, and socket glia and hypodermal cells (also known as epidermal cells) at the anterior tip of the animal. In addition to its cell-autonomous function, the non-cell-autonomous function of AJM-1 in glial and hypodermal cells is required for the specification of the AWCON subtype. Furthermore, we identified a role for the DEL-1 mechanosensitive DEG/ENaC channel in the calcium signaling pathway, mediated by UNC-2 and EGL-19 voltage-activated calcium channels, that specifies AWCOFF. Together, our results suggest a mechanism in which AJM-1 promotes SLO-1 expression and antagonizes mechanosensitive calcium signaling, thereby promoting the AWCON subtype. This study provides insight into the role of mechanical force in the stochastic lateralization of olfactory neurons.

SNED1 fibrillar assembly in the extracellular matrix requires fibronectin and collagen I

Leverton, L. — 2026-03-17

The extracellular matrix (ECM) is a meshwork of proteins that orchestrates a broad range of cellular phenotypes, including proliferation, adhesion, migration, and differentiation. SNED1 is a newly characterized ECM glycoprotein that promotes cell adhesion and is essential for embryonic development. Its upregulation is also associated with breast cancer metastasis and poor prognosis for breast cancer patients. We recently showed that SNED1 assembles into fibrillar structures, but the mechanisms guiding its incorporation into the ECM scaffold remain unknown. Combining biochemical assays and confocal immunofluorescence imaging, we found that SNED1 assembly in the ECM occurs early in the process of ECM building and is concomitant and overlaps with the deposition of fibronectin and collagen I, two major ECM proteins. By knocking down fibronectin or destabilizing collagen I fibers, we further demonstrate that SNED1 requires the presence of these proteins for its assembly. Last, using biolayer interferometry, we identify collagen I as the first direct binding partner of SNED1. Altogether, our results lay the foundation for future studies aimed at determining the mechanisms by which SNED1 fibers contribute to SNED1 pathophysiological functions. SUMMARY STATEMENTThe novel protein SNED1 requires the presence of fibronectin and collagen I to assemble into fibrillar structures in the extracellular matrix scaffold.

SNED1 modulates ECM architecture and cell proliferation via LDV-binding integrins

Pally, D. — 2026-03-17

The extracellular matrix (ECM) is a complex scaffold of proteins that supports multicellular structures. Interactions between cells and the ECM via receptors, like integrins, govern cellular phenotypes (e.g., proliferation, adhesion), but also contribute to ECM assembly. Understanding how ECM-receptor interactions regulate matrix assembly is critical to uncover how alterations of the ECM cause or accompany congenital diseases, cancer, or fibrosis. SNED1 is a novel ECM protein with roles in development and metastasis. However, the mechanisms governing its assembly and signaling functions remain largely unknown. SNED1 contains two integrin-binding motifs, RGD and LDV, and we recently showed that its interaction with RGD-integrins mediates cell adhesion. Here, we investigated the role of SNED1/integrin interactions in SNED1 ECM assembly. While SNED1/integrin interactions were not necessary for its initial incorporation in the ECM, interaction with LDV-, but not RGD-, integrins, was required for ECM build-up and the patterning of SNED1 and the fibrillar proteins fibronectin and collagen I. Moreover, SNED1/LDV-integrin interaction promoted ECM alignment, cell alignment, and cell proliferation, processes essential to SNED1-driven neural crest cell migration during craniofacial development and breast cancer invasion. SUMMARY STATEMENTInteraction of SNED1 with LDV-binding integrins, but not RGD-binding integrins, mediates ECM remodeling and controls cytoskeletal rearrangement and cell proliferation.

Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

Roach, S. T. — 2026-03-18

The formation of the premetastatic niche prepares distant tissues for tumor cell engraftment. Endothelial cells are critical mediators of premetastatic niche formation, orchestrating extravasation of circulating tumor cells and critical pro-tumor immune cells, such as neutrophils. In mouse models of breast cancer, we show that primary tumors upregulate the non-signaling chemokine receptor ACKR1 in the endothelium of the lung premetastatic niche. ACKR1-expressing venules were found to be preferential sites of neutrophil and tumor cell localization within lung tissue. A newly generated conditional ACKR1 allele was used to show that endothelial-specific removal of ACKR1 expression significantly reduces metastatic engraftment in the lung. When ACKR1 is activated by tumor-secreted factors, endothelial ACKR1 functions to promote neutrophil recruitment within the lung parenchyma. We conclude that ACKR1 is a critical component of the endothelial response to tumors at the metastatic site of the lung, leading to neutrophil recruitment and promotion of tumor cell metastasis. SUMMARYEndothelial cells play critical roles in breast cancer metastasis. ACKR1 is upregulated in the endothelium of the lung metastatic niche in response to primary mammary tumors. Endothelial ACKR1 expression was found to promote neutrophil infiltration into the metastatic niche and support breast tumor cell metastasis to the lung.

Functional bipartition of medial prefrontal cortex into salience detection and movement gain

Hou, S. — 2026-03-20

Reward prediction error (RPE) is a key function putatively realized by brain-wide neural states to drive learning and adaptive responding. The medial prefrontal cortex (mPFC) is among the many areas implicated in RPE computation. Yet, whether mPFC RPE processing differs as a function of valence, value, and sign is not well-understood. Here, we performed in vivo mPFC calcium imaging in rats engaged in an appetitive or aversive Pavlovian task designed to elicit RPE, along with continuous, unbiased behavioral monitoring. We found that short-latency bulk mPFC activity encodes salience independent of valence, value, or RPE. Surprisingly, as subsequently validated with in vivo bidirectional optogenetics, we show that salience-null mPFC activity modulates generalized movement in a valence- and task-agnostic fashion. Together, these results challenge region-wide RPE encoding by the mPFC, and instead, support a functional bipartition schema combining reactive detection of integrated salience and dynamic modulation of movement gain.

PPARγ-dependent and -independent regulation of methionine metabolism by diet-induced obesity and fasting in male mice.

Hawro, I. — 2026-03-27

Metabolic dysfunction-associated steatohepatitis (MASH) is associated with increased expression of peroxisome proliferator-activated receptor gamma (PPAR{gamma}, Pparg) and reduced expression of genes involved in methionine metabolism in the liver. The nuclear receptor PPAR{gamma} is activated by fatty acids, and the knockout of Pparg in hepatocytes (Pparg{Delta}Hep) reduced the negative effects of MASH on methionine metabolism. Here, we sought to determine whether hepatocyte Pparg is required for the transcriptional regulation of genes involved in hepatic methionine metabolism in conditions with altered fatty acid flux to the liver: fasting, refeeding, and high-fat diet (HFD)-induced obesity/steatosis. Fasting induced liver steatosis and increased the expression of key genes involved in the methionine metabolism in the liver, while 6h-refeeding reversed these effects and reduced the expression of phosphatidylethanolamine N-methyltransferase (Pemt) and cystathionine beta synthase (Cbs). Overall, fasting and refeeding did not alter hepatocyte Pparg expression nor Pparg{Delta}Hep affected fasting and refeeding-mediated regulation of methionine metabolism gene expression. Diet-induced steatosis reduced hepatic Pemt expression in control (Pparg-intact) mice, and the thiazolidinedione (TZD)-mediated activation of PPAR{gamma} in diet-induced obese control (Pparg-intact) mice reduced the expression of betaine homocysteine S-methyltransferase (Bhmt) and Cbs. However, diet-induced steatosis increased hepatocyte Pparg expression, and Pparg{Delta}Hep blocked the negative effects of HFD and TZD on hepatic methionine metabolism. The PPAR{gamma}-dependent reduction of hepatic Bhmt and Cbs expression was confirmed in mouse primary hepatocytes. Taken together, hepatocyte Pparg may serve as a negative regulator of hepatic methionine metabolism in diet-induced obese mice and these actions could contribute to promoting the onset of MASH.

A Wnt-responsive fibrocartilage progenitor system coordinates postnatal mandibular condylar cartilage growth

Inubushi, T. — 2026-03-27

Postnatal growth of the mandibular condyle requires coordinated expansion of fibrocartilage and production of chondrocytes, yet the cellular populations that organize this process remain incompletely defined. Here we identify a Wnt-responsive fibrocartilage progenitor population that contributes to postnatal mandibular condylar cartilage growth. Using a direct Wnt activity reporter (R26-WntVis), inducible genetic lineage tracing (Axin2CreERT2), and single-cell transcriptomics, we define a Wnt-enriched progenitor-like cluster localized predominantly within the fibrocartilage zone. Lineage tracing demonstrates that Axin2-lineage cells expand laterally within fibrocartilage and generate vertically aligned chondrocytes in the chondrocartilage compartment, indicating bidirectional growth contribution in vivo. Conditional ablation of {beta}-catenin in Axin2-lineage cells results in depletion of the fibrocartilage compartment and premature activation of chondrogenic differentiation programs, whereas constitutive {beta}-catenin activation disrupts compartmental organization without enhancing proliferation. Mechanistically, we identify Foxm1 as a Wnt-associated proliferative mediator enriched in fibrocartilage, and genetic reduction of Foxm1 cooperates with {beta}-catenin deficiency to impair condylar growth. In parallel, {beta}-catenin loss derepresses TGF-{beta}-Smad signaling and enhances chondrogenic differentiation, indicating that canonical Wnt activity coordinates proliferative maintenance while restraining lineage commitment within the same cellular compartment. Together, these findings identify a Wnt-responsive fibrocartilage progenitor system that regulates postnatal mandibular condylar cartilage growth by coupling Foxm1-associated proliferative maintenance with suppression of TGF-{beta}-dependent chondrogenic differentiation during temporomandibular joint development. Graphical abstractWnt-responsive fibrocartilage progenitors coordinate postnatal mandibular condylar cartilage growth through Foxm1-dependent proliferative maintenance and suppression of TGF-{beta}-driven chondrogenic differentiation.

Ubiquitin ligase CHFR impairs Tie2 signaling via K48-linked ubiquitylation and degradation of Akt1 in endothelial cells

Tiruppathi, C. — 2026-03-31

Vascular endothelial (VE)-cadherin is essential for maintaining endothelial junctional barrier integrity. The Angiopoietin-1 (Ang-1)/Tie2 axis induced Akt1 activation is crucial for maintaining endothelial junctional barrier by inhibiting FoxO1 and suppressing expression of Angiopoietin-2 (Ang-2), a Tie2 antagonist. Systemic inflammatory conditions such as sepsis, Akt1 expression is reduced, whereas FoxO1-dependent Ang-2 expression is increased, resulting in endothelial barrier dysfunction. We previously showed that the TLR4/FoxO1 axis induces the ubiquitin E3 ligase CHFR, which promotes endothelial barrier disruption by targeting VE-cadherin for ubiquitylation and degradation. However, little is known about Akt1 expression during vascular inflammation. Here, we identified FoxO1-dependent CHFR expression as a key mechanism driving K48-linked polyubiquitylation and proteasomal degradation of Akt1 in endothelial cells (EC). LPS-induced K48-linked ubiquitylation of Akt1 was prevented in CHFR-depleted human EC and in endothelial-specific Chfr knockout (Chfr{Delta}EC) mice. Accordingly, CHFR depletion increased Akt1 and VE-cadherin expression in both human lung EC and Chfr{Delta}EC mice. Chfr{Delta}EC mouse lungs also exhibited elevated Ang-1 and Tie2 expression, and Ang-1 stimulation induced sustained Akt1 phosphorylation in CHFR-deficient EC. Moreover, CHFR depletion prevented LPS-induced expression of FoxO1 and Ang-2 in EC. Mechanistically, CHFR interacted with phosphorylated Akt1 and mediated its ubiquitylation at lysine residues K30, K39, K154, and K268. Expression of a ubiquitylation-deficient Akt1 mutant prevented LPS-induced VE-cadherin degradation and vascular injury. Collectively, these findings identify CHFR as a critical regulator of endothelial inflammatory responses by controlling Akt1 stability and VE-cadherin expression during inflammation.

FAM134B isoform 2/RETREG1-2 defines a calnexin-TOLLIP-coupled ER-phagy pathway that restricts Ebola virus glycoprotein and is antagonized by VP40 through macro-autophagy

Zhang, J. — 2026-04-02

Selective autophagy of the endoplasmic reticulum (ER-phagy) is critical for ER proteostasis and host defense, yet how ER quality-control pathways interface with ER-phagy to restrict viral glycoproteins remains poorly defined. Previously, the 1st known ER-phagy receptor gene RETREG1 (RETR1)/FAM134B gene was reported to restrict Ebola virus (EBOV) replication in vivo by inhibiting the viral glycoprotein (GP) and viral protein 40 kDa (VP40) expression, but this mechanism remains unknown. Here, we identify the truncated RETR1/FAM134B isoform 2 (RETR1-2), but not its full-length protein RETR1, as an ER-phagy receptor that targets EBOV-GP for degradation. RETR1-2 broadly triggers GP degradation across ebolavirus species but not Marburg virus and inhibits EBOV replication. Mechanistically, RETR1-2 recognizes EBOV-GP via its luminal domain, undergoes GP-induced oligomerization, and directs GP-containing ER membranes to lysosomes through canonical macro-autophagy. Using unbiased mass spectrometry, we identified TOLLIP as the key cytoplasm adaptor for RETR1-2, which also requires cooperation with the ER chaperone calnexin for EBOV-GP degradation. Notably, the PI3P-binding C2 domain of TOLLIP mediates its interaction with RETR1-2, and the EBOV-GP degradation occurs independently of ubiquitination, revealing an unexpected role for TOLLIP in ER-phagy. Furthermore, EBOV-VP40 antagonizes this pathway by selectively targeting RETR1-2 for macroautophagic degradation independently of TOLLIP, thereby restoring GP expression and viral infectivity. Nevertheless, RETR1-2 reciprocally degrades VP40 via a similar mechanism. Together, these findings define a calnexin-TOLLIP-RETR1-2 axis that links ER quality control to ER-phagy-mediated antiviral restriction and uncover a reciprocal host-virus arms race centered on selective macro-autophagy.

Tuning a light-regulated allosteric switch for enhanced temporal control of protein activity

Matsche, J. — 2026-04-02

Optogenetics enables researchers to control protein localization, interactions, and activity using photosensitive domains. The key desired properties for optogenetic tools include broad applicability, tight light-regulated control with high dynamic range, and tunability. Previously, we described an engineered light-sensitive switch, LightR, composed of two VVD domains connected by a flexible linker, enabling light-dependent allosteric control of protein activity through site-specific insertion. Here, we introduce enhanced LightR variants with improved dynamic range and faster activation kinetics. Through targeted modifications to the VVD domains and linker region, we optimized a LightR-regulated Src kinase (LightR-Src) activity and generated two LightR-Src variants: one supporting sustained Src activation comparable to constitutively active Src, and another enabling rapid, reversible control, ideal for modeling transient signaling events suitable to mimic Src signaling in living cells. These modifications expand the versatility of LightR-based tools, facilitating their use in diverse optogenetic applications requiring high dynamic range of regulation and fast control of targeted proteins.

Unpredictable intermittent access exacerbates loss of control over ethanol drinking

Mitten, E. H. — 2026-04-03

BackgroundLoss of control over drinking is a hallmark feature of alcohol use disorder (AUD) that is modeled preclinically through escalation of ethanol consumption and aversion-resistant drinking. Prior work with other reinforcers suggests that within-session unpredictable, intermittent access (uIntA) promotes loss of control over intake. However, the effect of uIntA on voluntary ethanol consumption is unknown. MethodsMale and female Long-Evans rats (n=9-10/group) underwent seven weeks of daily voluntary ethanol (20% v/v) drinking sessions under either a continuous access (ContA) or uIntA schedule. Following four weeks of baseline, rats were rendered dependent using a two-week chronic intermittent ethanol vapor exposure procedure. Daily testing was maintained through one week into withdrawal from vapor exposure. On the final day of testing, ethanol was adulterated with quinine (30 mg/L) to assess aversion-resistant drinking. ResultsRats drinking under ContA and uIntA exhibited similar levels of average daily ethanol consumption at baseline. However, uIntA elicited a more robust dependence-induced escalation of ethanol consumption compared to ContA, with uIntA sustaining escalation through early protracted withdrawal. Additionally, while rats with ContA to ethanol remained sensitive to quinine even after chronic ethanol vapor exposure, uIntA promoted aversion-resistant drinking in ethanol dependent rats. ConclusionsThese results demonstrate that, compared to ContA, uIntA maintains ethanol drinking and exacerbates AUD-related symptomatology while also providing researchers with the ability to capture additional measures of motivation and drinking patterns without increasing experimental burden. This work positions uIntA as a powerful tool to assess psychological and neurobiological factors underlying loss of control over drinking.

A Tissue Virus Microenvironment with Activated Stress Responses Underlies Durable SIV Persistence

Hope, T. J. — 2026-04-03

HIV persistence during suppressive antiretroviral therapy (ART) remains a central barrier to cure, with the majority of reservoirs residing in gut-associated lymphoid tissues (GALT). Here, we define a spatially organized viral microenvironment (VME) that sustains reservoir durability and governs early viral rebound by comparing animals initiating ART early after infection (transient reservoirs) versus late (persistent reservoirs). Using immunoPET/CT-guided sampling of SIV-infected rhesus macaques combined with spatial transcriptomics, we interrogated tissue sites of viral production during the eclipse phase following analytical treatment interruption (ATI). Our results revealed that viral rebound from persistent reservoirs arises from discrete, transcriptionally active foci enriched in the mucosa lining the gut lumen. Eclipse phase persistent reservoirs were characterized by increased proviral burden and a distinct tissue state marked by activation of stress-response, metabolic, mitochondrial, and cell cycle programs coupled to repression of cytoplasmic translation and increased cellular senescence. These features co-occurred with immunosuppressive cellular architectures resembling tertiary lymphoid structures enriched for Treg cells, innate lymphoid cells, and mast cells, regulated by Treg-centered cell-cell interaction networks. In contrast, transient reservoirs displayed enhanced translational and metabolic activity and were embedded within immune-active environments enriched for CD8 T cells, Th17, Tfh, and activated CD4 T cells. Machine learning identified stress adaptation, hypoxia, metabolic rewiring, and cytoskeletal remodeling pathways as dominant predictors of viral density within persistent VMEs, with strong convergence on programs observed in tumor microenvironments (TME). Orthogonal validation confirmed activation of the integrated stress response (ISR) at sites of viral production in concurrence with results of immunofluorescent microscopy revealing SIV gag expression in two populations primarily in the mucosa, differentiated by the phosphorylation of eIF2. Together, these findings establish the VME as a critical determinant of reservoir persistence, integrating immune regulation, tissue remodeling, and translational control to enable viral survival. This framework suggests that effective HIV cure strategies will require coordinated disruption of VME-supportive functions in addition to targeting infected cells.