bioRxiv Channel: University of Connecticut Health Center

The maximum solubility product marks the threshold for condensation of multivalent biomolecules

Chattaraj, A. — 2022-10-05

Clustering of weakly interacting multivalent biomolecules underlies the formation of membraneless compartments known as condensates. As opposed to single component (homotypic) systems, the concentration dependence of multi-component (heterotypic) condensate formation is not well understood. We previously proposed the solubility product (SP), the product of monomer concentrations in the dilute phase, as a tool for understanding the concentration dependence of multi-component systems. In the current study, we further explore the limits of the SP concept using spatial Langevin dynamics and rule-based stochastic simulations. We show, for a variety of idealized molecular structures, how the maximum SP coincides with the onset of the phase transition, i.e., the formation of large clusters. We reveal the importance of intra-cluster binding in steering the free and cluster phase molecular distributions. We also show how structural features of biomolecules shape the solubility product profiles. The interplay of flexibility, length and steric hindrance of linker regions controls the phase transition threshold. Remarkably, when solubility products are normalized to non-dimensional variables and plotted against the concentration scaled to the threshold for phase transition, the curves all coincide independent of the structural features of the binding partners. Similar coincidence is observed for the normalized clustering vs. concentration plots. Overall, the principles derived from these systematic models will help guide and interpret in vitro and in vivo experiments on the biophysics of biomolecular condensates. Significance StatementBiomolecular condensates are macroscopic intracellular structures that are composed of weakly interacting macromolecules. Because their composition can be complex, there are no simple rules for how condensates form as a function of the concentrations of their individual components. In this work, we show how the solubility product (SP), the product of monomer concentrations in the dilute phase, might serve as a tool for predicting the concentration dependence for condensation of multi-component systems. Specifically, Langevin dynamics simulations of the clustering of a series of multivalent binding partners reveals how the maximum SP is always attained at the same concentration as the appearance of large clusters. Experimental application of the SP concept should help rationalize the cellular formation of biomolecular condensates.

Recombinant Human Proteoglycan 4 (rhPRG4) Downregulates TNFα-Stimulated NFκB Activity and FAT10 Expression in Human Corneal Epithelial Cells

Menon, N. G. — 2022-10-12

Dry Eye Disease (DED) is a complex pathology affecting millions of people with significant impact on quality of life. Corneal inflammation, including via the NF{kappa}B pathway, plays a key etiological role in DED. Recombinant human proteoglycan 4 (rhPRG4) has been shown to be a clinically effective treatment for DED that has anti-inflammatory effects in corneal epithelial cells, but the underlying mechanism is still not understood. Our goal was to understand if rhPRG4 affects TNF-stimulated inflammatory activity in corneal epithelial cells. We treated hTERT-immortalized corneal epithelial (hTCEpi) cells {+/-}TNF {+/-}rhPRG4 and performed Western blotting on cell lysate and RNA sequencing. Bioinformatics analysis revealed that rhPRG4 had a significant effect on TNF-mediated inflammation with potential effects on matricellular homeostasis. rhPRG4 reduced activation of key inflammatory pathways and decreased expression of transcripts for key inflammatory cytokines, interferons, interleu-kins, and transcription factors. TNF treatment significantly increased phosphorylation and nuclear translocation of p65, and rhPRG4 significantly reduced both these effects. RNA sequencing identified FAT10, which has not been studied in the context of DED, as a key pro-inflammatory transcript increased by TNF and decreased by rhPRG4. These results were confirmed at the protein level. In summary, rhPRG4 is able to downregulate NF{kappa}B activity in hTCEpi cells, suggesting a potential biological mechanism by which it may act as a therapeutic for DED.

Modeling the iron storage protein ferritin reveals how residual ferrihydrite iron determines initial ferritin iron sequestration kinetics

Masison, J. — 2022-11-04

Ferritin is the primary iron storage protein in mammalian cells and is necessary for cellular iron homeostasis. Its ability to sequester iron protects cells from rising concentrations of ferrous iron limiting oxidative cell damage. The dynamics of its sequestration behavior have been studied extensively experimentally, but mostly in vitro, rather than within mammalian cells, computational modeling efforts are lacking, and several open questions remain. The importance of FT modeling is driven by the idea that understanding the kinetics of ferritin iron sequestration and how it impacts the regulation of iron metabolism in the cells that compose the main iron regulatory tissues (eg. Kupffer cells, splenic macrophages, enterocytes, erythroblasts, etc.) is central to understanding systemic iron control. The focus of the work here is establishing a model that tractably simulates the ferritin iron sequestration kinetics in a mechanistic way for incorporation into larger cell models, in addition to contributing to the understanding of ferritin iron sequestration dynamics within cells. The models parameter values were determined from published kinetic and binding experiments and the model was validated against independent data not used in its construction. Simulation results indicate that FT concentration is the most impactful on overall sequestration dynamics, while the FT iron saturation (number of iron atoms sequestered per FT cage) fine tunes the initial rates. Finally, because this model has a small number of reactions and species, was built to represent important mechanistic details of FT kinetics, and has flexibility to include subtle changes in subunit composition, we propose this model to be used as a building block for models of iron metabolism in a variety of specific cell types.

Homophilic interaction of E-Cadherin prevents cell-cell fusion between developing germline and surrounding epithelia in Drosophila ovary

Antel, M. — 2022-11-23

In the Drosophila ovary, developing germline cysts are encapsulated by somatic follicle cell epithelia and E-Cadherin localizes to the interface of these tissues. E-Cadherin mutants have been shown to have multiple defects in oogenesis. Therefore, it is difficult to determine E-Cadherin function on germline-soma interaction. In this study, we characterize E-Cadherin function, specifically focusing on germline-soma interaction. Unexpectedly, knockdown of E-Cadherin either in the germline or follicle cells results in excess formation of membrane protrusions at the interface of these cells, which leads to a cell-cell fusion and indicates that homophilic interaction of E-Cadherin is required for maintenance of the tissue boundary between these two adjacent tissues. The fate of follicle cells fused to the germline becomes compromised, leading to a defective individualization of germline cysts. We propose that homophilic interaction of E-Cadherin facilitates a barrier between adjacent tissues, demonstrating a unique model of cell-fate disturbance caused by cell-cell fusion.

Centromere-associated retroelement evolution in Drosophila melanogaster reveals an underlying conflict

Hemmer, L. W. — 2022-11-26

Centromeres are chromosomal regions essential for coordinating chromosome segregation during cell division. While centromeres are defined by the presence of a centromere-specific histone H3 variant rather than a particular DNA sequence, they are typically embedded in repeat-dense chromosomal genome regions. In many species, centromeres are associated with transposable elements, but it is unclear if these elements are selfish or if they play a role in centromere specification or function. Here we use Drosophila melanogaster as a model to understand the evolution of centromere-associated transposable elements. G2/Jockey-3 is a non-LTR retroelement in the Jockey clade and the only sequence shared by all centromeres. We study the evolution of G2/Jockey-3 using short and long read population genomic data to infer insertion polymorphisms across the genome. We combine estimates of the age, frequency, and location of insertions to infer the evolutionary processes shaping G2/Jockey-3 and its association with the centromeres. We find that G2/Jockey-3 is an active retroelement targeted by the piRNA pathway that is enriched in centromeres at least in part due to an insertion bias. We do not detect signatures of positive selection on any G2/Jockey-3 insertions that would suggest than individual copies are favored by natural selection. Instead, we infer that most insertions are neutral or weakly deleterious both inside and outside of the centromeres. Therefore, G2/Jockey-3 evolution is consistent with it being a selfish genetic element that targets centromeres. We propose that targeting centromeres helps active retroelements escape host defenses, as the unique centromeric chromatin may prevent targeting by the host silencing machinery. At the same time, centromeric TEs insertions may be tolerated or even beneficial if they also contribute to the transcriptional and chromatin environment. Thus, we suspect centromere-associated retroelements like G2/Jockey-3 reflect a balance between conflict and cooperation at the centromeres.

The microbial community dynamics of cocaine sensitization in two behaviorally divergent strains of collaborative cross mice

Tran, T. D. B. — 2022-12-07

The gut-brain axis is increasingly recognized as an important pathway involved in cocaine use disorder. Microbial products of the murine gut have been shown to affect striatal gene expression, and depletion of the microbiome by antibiotic treatment alters cocaine-induced behavioral sensitization in C57BL/6J male mice. Some reports suggest that cocaine-induced behavioral sensitization is correlated with drug self-administration behavior in mice. Here we profile the composition of the naive microbiome and its response to cocaine sensitization in two Collaborative Cross (CC) strains. These strains display extremely divergent behavioral responses to cocaine sensitization. A high-responding strain, CC004/TauUncJ (CC04), has a gut microbiome that contains a greater amount of Lactobacillus than the cocaine-nonresponsive strain CC041/TauUncJ (CC41). The gut microbiome of CC41 is characterized by an abundance of Eisenbergella, Robinsonella and Ruminococcus. In response to cocaine, CC04 has an increased Barnsiella population, while the gut microbiome of CC41 displays no significant changes. PICRUSt functional analysis of the functional potential of the gut microbiome in CC04 shows a significant number of potential gut-brain modules altered after exposure to cocaine, specifically those encoding for tryptophan synthesis, glutamine metabolism, and menaquinone synthesis (vitamin K2). Depletion of the microbiome by antibiotic treatment revealed an altered cocaine-sensitization response following antibiotics in female CC04 mice. Depleting the microbiome by antibiotic treatment in males revealed increased infusions for CC04 during a cocaine intravenous self-administration dose-response curve. Together these data suggest that genetic differences in cocaine-related behaviors may involve the microbiome. FUNDINGU01DA043809 to JAB, GMW P50DA039841 to EJC

ModelCIF: An extension of PDBx/mmCIF data representation for computed structure models

Vallat, B. — 2022-12-09

ModelCIF (github.com/ihmwg/ModelCIF) is a data information framework developed for and by computational structural biologists to enable delivery of Findable, Accessible, Interoperable, and Reusable (FAIR) data to users worldwide. It is an extension of the Protein Data Bank Exchange / macromolecular Crystallographic Information Framework (PDBx/mmCIF), which is the global data standard for representing experimentally-determined, three-dimensional (3D) structures of macromolecules and associated metadata. ModelCIF provides an extensible data representation for deposition, archiving, and public dissemination of predicted 3D models of proteins. The PDBx/mmCIF framework and its extensions (e.g., ModelCIF) are managed by the Worldwide Protein Data Bank partnership (wwPDB, wwpdb.org) in collaboration with relevant community stakeholders such as the wwPDB ModelCIF Working Group (wwpdb.org/task/modelcif). This semantically rich and extensible data framework for representing computed structure models (CSMs) accelerates the pace of scientific discovery. Herein, we describe the architecture, contents, and governance of ModelCIF, and tools and processes for maintaining and extending the data standard. Community tools and software libraries that support ModelCIF are also described.

Wnt-associated adult stem cell marker Lgr6 is required for osteogenesis and fracture healing

Doherty, L. — 2022-12-10

Despite the remarkable regenerative capacity of skeletal tissues, nonunion of bone and failure of fractures to heal properly presents a significant clinical concern. Stem and progenitor cells are present in bone and become activated following injury; thus, elucidating mechanisms that promote adult stem cell-mediated healing is important. Wnt-associated adult stem marker Lgr6 is implicated in the regeneration of tissues with well-defined stem cell niches in stem cell-reliant organs. Here, we demonstrate that Lgr6 is dynamically expressed in osteoprogenitors in response to fracture injury. Using an Lgr6-null mouse model, we find that Lgr6 expression is necessary for maintaining bone volume and efficient postnatal bone regeneration in adult mice. Skeletal progenitors isolated from Lgr6-null mice have reduced colony-forming potential and reduced osteogenic differentiation capacity due to attenuated cWnt signaling. Lgr6-null mice consist of a lower proportion of self-renewing stem cells. In response to fracture injury, Lgr6-null mice have deficient proliferation of periosteal progenitors and reduced ALP activity. Further, analysis of bone regeneration phase and remodeling phase of fracture healing in Lgr6-null mice showed impaired endochondral ossification and reduced mineralization. We propose that in contrast to not being required for successful skeletal development Lgr6-positive cells have a direct role in endochondral bone repair.

Correction of transposase sequence bias in ATAC-seq data with rule ensemble modeling

Wolpe, J. B. — 2022-12-12

Chromatin accessibility assays have revolutionized the field of transcription regulation by providing single-nucleotide resolution measurements of regulatory features such as promoters and transcription factor binding sites. ATAC-seq directly measures how well the Tn5 transpose accesses chromatinized DNA. Tn5 has a complex sequence bias that is not effectively scaled with traditional bias-correction methods. We model this complex bias using a rule ensemble machine learning approach that integrates information from many input k-mers proximal to the ATAC sequence reads. We effectively characterize and correct single-nucleotide sequence biases and regional sequence biases of the Tn5 enzyme. Correction of enzymatic sequence bias is an important step in interpreting chromatin accessibility assays that aim to infer transcription factor binding and regulatory activity of elements in the genome.

Processing and evaluating the quality of genome-wide nascent transcription profiling libraries

Scott, T. G. — 2022-12-16

Precision genomic run-on assays (PRO-seq) quantify nascent RNA at single nucleotide resolution with strand specificity. Here we deconstruct a recently published genomic nascent RNA processing pipeline (PEPPRO) into its components and link the analyses to the underlying molecular biology. PRO-seq experiments are evolving and variations can be found throughout the literature. The analyses are presented as individual code chunks with comprehensive details so that users can modify the framework to accommodate different protocols. We present the framework to quantify the following quality control metrics: library complexity, nascent RNA purity, nuclear run-on efficiency, alignment rate, sequencing depth, and RNA degradation.

Predicting alcohol-related memory problems in older adults: A machine learning study with multi-domain features

Kamarajan, C. — 2023-01-02

Memory problems are common among older adults with a history of alcohol use disorder (AUD). Employing a machine learning framework, the current study investigates the use of multi-domain features to classify individuals with and without alcohol-induced memory problems. A group of 94 individuals (ages 50-81 years) with alcohol-induced memory problems (Memory group) were compared with a matched Control group who did not have memory problems. The Random Forests model identified specific features from each domain that contributed to the classification of Memory vs. Control group (AUC=88.29%). Specifically, individuals from the Memory group manifested a predominant pattern of hyperconnectivity across the default mode network regions except some connections involving anterior cingulate cortex which were predominantly hypoconnected. Other significant contributing features were (i) polygenic risk scores for AUD, (ii) alcohol consumption and related health consequences during the past 5 years, such as health problems, past negative experiences, withdrawal symptoms, and the largest number of drinks in a day during the past 12 months, and (iii) elevated neuroticism and increased harm avoidance, and fewer positive "uplift" life events. At the neural systems level, hyperconnectivity across the default mode network regions, including the connections across the hippocampal hub regions, in individuals with memory problems may indicate dysregulation in neural information processing. Overall, the study outlines the importance of utilizing multidomain features, consisting of resting-state brain connectivity collected [~]18 years ago, together with personality, life experiences, polygenic risk, and alcohol consumption and related consequences, to predict alcohol-related memory problems that arise in later life.

Calponin 2 harnesses metabolic reprogramming to determine kidney fibrosis

Gui, Y. — 2023-01-03

In the fibrotic kidneys, the extent of a formed deleterious microenvironment is determined by cellular mechanical forces. This process requires metabolism for energy; however, how cellular mechanics and metabolism are connected remains unclear. Our proteomics revealed that actin filament binding and cell metabolism are the two most dysregulated events in the fibrotic kidneys. As a prominent actin stabilizer, Calponin 2 (CNN2) is predominantly expressed in fibroblasts and pericytes. CNN2 knockdown preserves kidney function and alleviates fibrosis. Global proteomics profiled that CNN2 knockdown enhanced the activities of the key rate-limiting enzymes and regulators of fatty acid oxidation (FAO) in diseased kidneys. Inhibiting carnitine palmitoyltransferase 1 in the FAO pathway results in lipid accumulation and extracellular matrix deposition in the fibrotic kidneys, which were restored after CNN2 knockdown. In patients, increased serum CNN2 levels are correlated with lipid content. Bioinformatics and chromatin immunoprecipitation showed that CNN2 interactor, estrogen receptor 2 (ESR2) binds peroxisome proliferator-activated receptor- (PPAR) to transcriptionally regulate FAO downstream target genes expression amid kidney fibrosis. In vitro, ESR2 knockdown repressed the mRNA levels of PPAR and the key genes in the FAO pathway. Conversely, activation of PPAR reduced CNN2-induced matrix inductions. Our results suggest that balancing cell mechanics and metabolism is crucial to develop therapeutic strategies to halt kidney fibrosis.

Activation Dynamics of Ubiquitin Specific Protease 7

Valles, G. J. — 2023-01-12

Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme responsible for the regulation of key human oncoproteins and tumor suppressors including Mdm2 and p53, respectively. Unlike other members of the USP family of proteases, the isolated catalytic domain of USP7 adopts an enzymatically inactive conformation that has been well characterized using X-ray crystallography. The catalytic domain also samples an active conformation, which has only been captured upon USP7 substrate-binding. Here, we utilized CPMG NMR relaxation dispersion studies to observe the dynamic motions of USP7 in solution. Our results reveal that the catalytic domain of USP7 exchanges between two distinct conformations, the inactive conformation populated at 95% and the active conformation at 5%. The largest structural changes are localized within functionally important regions of the enzyme including the active site, the ubiquitin-binding fingers, and the allosteric helix of the enzyme, suggesting that USP7 can adopt its active conformation in the absence of a substrate. Furthermore, we show that the allosteric L299A activating mutation disturbs this equilibrium, slows down the exchange, and increases the residence time of USP7 in its active conformation, thus, explaining the elevated activity of the mutant. Overall, this work shows that the isolated USP7 catalytic domain pre-samples its "invisible" active conformation in solution, which may contribute to its activation mechanism.

Structural insights into regulation of TRPM7 divalent cation uptake by the small GTPase ARL15

Mahbub, L. — 2023-01-20

Cystathionine-{beta}-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) are an evolutionarily conserved family of magnesium transporters. They promote efflux of Mg2+ ions on their own or uptake of divalent cations when coupled to the transient receptor potential ion channel subfamily M member 7 (TRPM7). Recently, ADP-ribosylation factor-like GTPase 15 (ARL15) has been identified as CNNM binding partner and an inhibitor of divalent cation influx by TRPM7. Here, we characterize ARL15 as a GTP-binding protein and demonstrate that it binds the CNNM CBS-pair domain with low micromolar affinity. The crystal structure of the complex between ARL15 GTPase domain and CNNM2 CBS-pair domain reveals the molecular determinants of the interaction and allowed the identification of mutations in ARL15 and CNNM2 mutations that abrogate binding. Loss of CNNM binding prevented ARL15 suppression of TRPM7 channel activity in support of previous reports that the proteins function as a ternary complex. Binding experiments with phosphatase of regenerating liver 2 (PRL2 or PTP4A2) revealed that ARL15 and PRLs compete for binding CNNM, suggesting antagonistic regulation of divalent cation transport by the two proteins.

Transcriptomic profiling of retinal cells reveals a subpopulation of microglia/macrophages expressing Rbpms and Spp1 markers of retinal ganglion cells (RGCs) that confound identification of RGCs

Theune, W. C. — 2023-01-24

Analysis of retinal ganglion cells (RGCs) by scRNA-seq is emerging as a state-of-the-art method for studying RGC biology and subtypes, as well as for studying the mechanisms of neuroprotection and axon regeneration in the central nervous system (CNS). Rbpms has been established as a pan-RGC marker, and Spp1 has been established as an RGC type marker. Here, we analyzed by scRNA-seq retinal microglia and macrophages, and found Rbpms+ and Spp1+ subpopulations of retinal microglia/macrophages, which pose a potential pitfall in scRNA-seq studies involving RGCs. We performed comparative analysis of cellular identity of the presumed RGC cells isolated in recent scRNA-seq studies, and found that Rbpms+ and Spp1+ microglia/macrophages confounded identification of RGCs. We also provide solutions for circumventing this potential pitfall in scRNA-seq studies, by including in RGC and RGC selection criteria other pan-RGC and RGC markers.

Investigating variant and expression of CVD genes associated phenotypes among high-risk Heart Failure patients

Ahmed, Z. — 2023-01-25

Cardiovascular disease (CVD) is a leading cause of premature mortality in the US and the world. CVD comprises of several complex and mostly heritable conditions, which range from myocardial infarction to congenital heart disease. Here, we report our findings from an integrative analysis of gene expression, disease-causing gene variants, and associated phenotypes among CVD populations, with a focus on high-risk Heart Failure (HF) patients. We built a cohort using electronic health records (EHR) of consented patients with available samples, and then performed high-throughput whole-genome and RNA sequencing (RNA-seq) of key genes responsible for HF and other CVD pathologies. We also incorporated a translational aspect to our study by integrating genomics findings with patient medical records. This involved linking ICD-10 codes with our gene expression and variant data to identify associations with HF and other CVDs. Our in-depth gene expression analysis revealed differentially expressed genes associated with HF (41 genes) and other CVDs (23 genes). Furthermore, a variant analysis of whole-genome sequence data of CVD patients identified genes with altered gene expression (FLNA, CST3, LGALS3, and HBA1) with functional and nonfunctional mutations in these genes. Our study highlights the importance of an integrative approach that leverages gene expression, genetic mutations, and clinical data that will allow the prioritization of key driver genes for complex diseases to improve personalized healthcare.

Astrocytic TIMP-1 regulates production of Anastellin, a novel inhibitor of oligodendrocyte differentiation and FTY720 responses

Sutter, P. A. — 2023-02-18

Astrocyte activation is associated with neuropathology and the production of tissue inhibitor of metalloproteinase-1 (TIMP1). TIMP1 is a pleiotropic extracellular protein that functions both as a protease inhibitor and as a growth factor. We have previously demonstrated that murine astrocytes that lack expression of Timp1 do not support rat oligodendrocyte progenitor cell (rOPC) differentiation, and adult global Timp1 knockout (Timp1KO) mice do not efficiently remyelinate following a demyelinating injury. To better understand the basis of this, we performed unbiased proteomic analyses and identified a fibronectin-derived peptide called anastellin that is unique to the murine Timp1KO astrocyte secretome. Anastellin was found to block rOPC differentiation in vitro and enhanced the inhibitory influence of fibronectin on rOPC differentiation. Anastellin is known to act upon the sphingosine-1-phosphate receptor 1 (S1PR1), and we determined that anastellin also blocked the pro-myelinating effect of FTY720 (or fingolimod) on rOPC differentiation in vitro. Further, administration of FTY720 to wild-type C57BL/6 mice during MOG35-55-EAE ameliorated clinical disability while FTY720 administered to mice lacking expression of Timp1 in astrocytes (Timp1cKO) had no effect. Analysis of human TIMP1 and fibronectin (FN1) transcripts from healthy and multiple sclerosis (MS) patient brain samples revealed an inverse relationship where lower TIMP1 expression was coincident with elevated FN1 in MS astrocytes. Lastly, we analyzed proteomic databases of MS samples and identified anastellin peptides to be more abundant in the cerebrospinal fluid (CSF) of human MS patients with high versus low disease activity. The prospective role for anastellin generation in association with myelin lesions as a consequence of a lack of astrocytic TIMP-1 production could influence both the efficacy of fingolimod responses and the innate remyelination potential of the the MS brain. Significance StatementAstrocytic production of TIMP-1 prevents the protein catabolism of fibronectin. In the absence of TIMP-1, fibronectin is further digested leading to a higher abundance of anastellin peptides that can bind to sphingosine-1-phosphate receptor 1. The binding of anastellin with the sphingosine-1-phosphate receptor 1 impairs the differentiation of oligodendrocytes progenitor cells into myelinating oligodendrocytes in vitro, and negates the astrocyte-mediated therapeutic effects of FTY720 in the EAE model of chronic CNS inflammation. These data indicate that TIMP-1 production by astrocytes is important in coordinating astrocytic functions during inflammation. In the absence of astrocyte produced TIMP-1, elevated expression of anastellin may represent a prospective biomarker for FTY720 therapeutic responsiveness.

Isogenic hiPSC models of Turner Syndrome reveal shared roles of inactive X and Y in the human cranial neural crest network

Ahern, D. T. — 2023-03-09

SUMMARYModeling the developmental etiology of viable human aneuploidy can be challenging in rodents due to syntenic boundaries, or primate-specific biology. In humans, monosomy-X (45,X) causes Turner syndrome (TS), altering craniofacial, skeletal, endocrine, and cardiovascular development, which in contrast remain unaffected in 39,X-mice. To learn how human monosomy-X may impact early embryonic development, we turned to human 45,X and isogenic euploid induced pluripotent stem cells (hiPSCs) from male and female mosaic donors. Because neural crest (NC) derived cell types are hypothesized to underpin craniofacial and cardiovascular changes in TS, we performed a highly-powered differential expression study on hiPSC-derived anterior neural crest cells (NCCs). Across three independent isogenic panels, 45,X NCCs show impaired acquisition of PAX7+SOX10+ markers, and disrupted expression of other NCC-specific genes, relative to their isogenic euploid controls. In particular, 45,X NCCs increase cholesterol biosynthesis genes while reducing transcripts that feature 5 terminal oligopyrimidine (TOP) motifs, including those of ribosomal protein and nuclear-encoded mitochondrial genes. Such metabolic pathways are also over-represented in weighted co-expression gene modules that are preserved in monogenic neurocristopathy. Importantly, these gene modules are also significantly enriched in 28% of all TS-associated terms of the human phenotype ontology. Our analysis identifies specific sex-linked genes that are expressed from two copies in euploid males and females alike and qualify as candidate haploinsufficient drivers of TS phenotypes in NC-derived lineages. This study demonstrates that isogenic hiPSC-derived NCC panels representing monosomy-X can serve as a powerful model of early NC development in TS and inform new hypotheses towards its etiology.

Pharmacologic Activation of an Integrated Stress Response Kinase Promotes Mitochondrial Remodeling

Perea, V. — 2023-03-12

The integrated stress response (ISR) comprises the eIF2 kinases PERK, GCN2, HRI, and PKR, which induce translational and transcriptional signaling in response to diverse insults. Deficiencies in PERK signaling lead to mitochondrial dysfunction and contribute to the pathogenesis of numerous diseases. We define the potential for pharmacologic activation of compensatory eIF2 kinases to rescue ISR signaling and promote mitochondrial adaptation in PERK-deficient cells. We show that the HRI activator BtdCPU and GCN2 activator halofuginone promote ISR signaling and rescue ER stress sensitivity in PERK-deficient cells. However, BtdCPU induces mitochondrial depolarization, leading to mitochondrial fragmentation and activation of the OMA1-DELE1-HRI signaling axis. In contrast, halofuginone promotes mitochondrial elongation and adaptive mitochondrial respiration, mimicking regulation induced by PERK. This shows halofuginone can compensate for deficiencies in PERK signaling and promote adaptive mitochondrial remodeling, highlighting the potential for pharmacologic ISR activation to mitigate mitochondrial dysfunction and motivating the pursuit of highly-selective ISR activators.

Cross-species single-cell comparison of systemic and cardiac inflammatory responses after cardiac injury

Cortada, E. — 2023-03-16

The immune system coordinates the response to cardiac injury and is known to control regenerative and fibrotic scar outcomes in the heart and subsequent chronic low-grade inflammation associated with heart failure. Here we profiled the inflammatory response to heart injury using single cell transcriptomics to compare and contrast two experimental models with disparate outcomes. We used adult mice, which like humans lack the ability to fully recover and zebrafish which spontaneously regenerate after heart injury. The extracardiac reaction to cardiomyocyte necrosis was also interrogated to assess the specific peripheral tissue and immune cell reaction to chronic stress. Cardiac macrophages are known to play a critical role in determining tissue homeostasis by healing versus scarring. We identified distinct transcriptional clusters of monocytes/macrophages in each species and found analogous pairs in zebrafish and mice. However, the reaction to myocardial injury was largely disparate between mice and zebrafish. The dichotomous response to heart damage between the mammalian and zebrafish monocytes/macrophages may underlie the impaired regenerative process in mice, representing a future therapeutic target.

Neuronal phase shifts differ for excitation vs. inhibition: a computer modeling study

Kelley, C. — 2023-03-21

Rhythmic activity is ubiquitous in neural systems, and impedance analysis has been widely used to examine frequency-dependent responses of neuronal membranes to rhythmic inputs. Impedance analysis assumes the neuronal membrane is a linear system, requiring the use of small signals to stay in a near-linear regime. However, postsynaptic potentials are often large and trigger nonlinear mechanisms. We therefore augmented impedance analysis to evaluate membrane responses in this nonlinear domain, analyzing responses to injected current for subthreshold membrane voltage (Vmemb), suprathreshold spike-blocked Vmemb, and spiking in a validated neocortical pyramidal neuron computer model. Responses in these output regimes were asymmetrical, with different phase shifts during hyperpolarizing and depolarizing half-cycles. Suprathreshold chirp stimulation gave equivocal results due to nonstationarity of response, requiring us to use fixed-frequency sinusoids. Sinusoidal inputs produced phase retreat: action potentials occurred progressively later in cycles of the input stimulus, resulting from adaptation. Conversely, sinusoidal current with increasing amplitude over cycles produced a pattern of phase advance: action potentials occurred progressively earlier. Phase retreat was dependent on Ih and IAHP currents; phase advance was modulated by these currents. Our results suggest differential responses of cortical neurons depending on the frequency of oscillatory input in the delta - beta range, which will play a role in neuronal responses to shifts in network state. We hypothesize that intrinsic cellular properties complement network properties and contribute to in vivo phase-shift phenomena such as phase precession, seen in place and grid cells, and phase roll, observed in hippocampal CA1 neurons. New & NoteworthyWe augmented electrical impedance analysis to characterize phase shifts between large amplitude current stimuli and nonlinear, asymmetric membrane potential responses. We predict different frequency-dependent phase shifts in response excitation versus inhibition, as well as shifts in spike timing over multiple input cycles, in resonant pyramidal neurons. We hypothesize that these effects contribute to navigation-related phenomena like phase precession and phase roll. Our neuron-level hypothesis complements, rather than falsifies, prior network-level explanations of these phenomena.

Coordination of Alternative Splicing and Alternative Polyadenylation revealed by Targeted Long Read Sequencing

Zhang, Z. — 2023-03-23

Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. Here, the coordination of cassette exon (CE) splicing and APA in Drosophila was investigated using a targeted long-read sequencing approach we call Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to resolve the connectivity of alternative exons to alternative 3 ends. Using PL-Seq, we identified genes that exhibit significant differences in CE splicing depending on connectivity to short versus long 3UTRs. Genomic long 3UTR deletion was found to alter upstream CE splicing in short 3UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3UTRs. This work highlights the importance of considering connectivity to alternative 3UTRs when monitoring AS events.

Cellular Senescence is a Double-Edged Sword in Regulating Aged Immune Responses to Influenza

Torrance, B. L. — 2023-04-11

Clearance of senescent cells has demonstrated therapeutic potential in the context of chronic age-related diseases. Little is known, however, how clearing senescent cells affects the ability to respond to an acute infection and form quality immunological memory. We aimed to probe the effects of clearing senescent cells in aged mice on the immune response to influenza (flu) infection. We utilized a p16 trimodality reporter mouse model (p16-3MR) to allow for identification and selective deletion of p16-expressing senescent cells upon administration of ganciclovir (GCV). While p16-expressing senescent cells may exacerbate dysfunctional responses to a primary infection, our data suggest they may play a role in fostering memory cell generation. We demonstrate that although deletion of p16-expressing cells enhanced viral clearance, this also severely limited antibody production in the lungs of flu-infected aged mice. 30 days later, there were fewer flu-specific CD8 memory T cells and lower levels of flu-specific antibodies in the lungs of GCV treated mice. GCV treated mice were unable to mount an optimal memory response and demonstrated increased viral load following a heterosubtypic challenge. These results suggest that targeting senescent cells may potentiate primary responses while limiting the ability to form durable and protective immune memory with age.

Luteinizing hormone stimulates ingression of granulosa cells within the mouse preovulatory follicle.

Owen, C. M. — 2023-04-21

Luteinizing hormone (LH) induces ovulation by acting on its receptors in the mural granulosa cells that surround a mammalian oocyte in an ovarian follicle. However, much remains unknown about how activation of the LH receptor modifies the structure of the follicle such that the oocyte is released and the follicle remnants are transformed into the corpus luteum. The present study shows that the preovulatory surge of LH stimulates LH receptor-expressing granulosa cells, initially located almost entirely in the outer layers of the mural granulosa, to rapidly extend inwards, intercalating between other cells. The cellular ingression begins within 30 minutes of the peak of the LH surge, and the proportion of LH receptor-expressing cell bodies in the inner half of the mural granulosa layer increases until the time of ovulation, which occurs at about 10 hours after the LH peak. During this time, many of the initially flask-shaped cells appear to detach from the basal lamina, acquiring a rounder shape with multiple filipodia. Starting at about 4 hours after the LH peak, the mural granulosa layer at the apical surface of the follicle where ovulation will occur begins to thin, and the basolateral surface develops invaginations and constrictions. Our findings raise the question of whether LH stimulation of granulosa cell ingression may contribute to these changes in the follicular structure that enable ovulation. Summary sentenceLH-induced ingression of LH receptor-expressing cells within the mural granulosa layer of the ovarian follicle is a new component in the complex sequence of structural changes that lead to ovulation.

Late- life shift in caloric intake affects fly longevity and metabolism

Li, M. — 2023-05-14

Caloric restriction (CR) delays the onset of age-related changes and extends lifespan in most species, but how late in life organisms benefit from switching to a low-calorie (L) diet is unexplored. We transferred wild type male flies from a high- (H) to a L-calorie diet (HL) or vice versa (LH) at different times. Late-life HL shift immediately and profoundly reduces fly mortality rate to briefly lower rate than in flies on a constant L diet, and increases lifespan. Conversely, a LH shift increases mortality and hazard rate, which is temporarily higher than in flies aged on a H diet, and leads to shorter lifespan. Transcriptomic changes within 48 hours following diet shift uncover physiological adaptations to available nutrients. Unexpectedly, more abundant transcriptomic changes accompanied LH shift, including ribosome biogenesis, and promotion of growth, which likely contributes to higher mortality rate. Considering that the beneficial effects of CR on physiology and lifespan are conserved across many organisms, our findings suggest that CR interventions in older humans may counteract the detrimental effects of H diets even when initiated later in life.

The androgen receptor does not directly regulate the transcription of DNA damage response genes

Hasterok, S. — 2023-05-14

The clinical success of combined androgen deprivation therapy (ADT) and radiation therapy (RT) in prostate cancer (PCa) created interest in understanding the mechanistic links between androgen receptor (AR) signaling and the DNA damage response (DDR). Convergent data have led to a model where AR both regulates, and is regulated by, the DDR. Integral to this model is that the AR regulates the transcription of DDR genes both at steady state and in response to ionizing radiation (IR). In this study, we sought to determine which immediate transcriptional changes are induced by IR in an AR-dependent manner. Using PRO-seq to quantify changes in nascent RNA transcription in response to IR, the AR antagonist enzalutamide, or the combination of the two, we find that enzalutamide treatment significantly decreased expression of canonical AR target genes but had no effect on DDR gene sets in PCa cells. Surprisingly, we also found that the AR is not a primary regulator of DDR genes either in response to IR or at steady state in asynchronously growing PCa cells. Our data indicate that the clinical benefit of ADT and RT is not due to the direct regulation of DDR gene transcription by AR.

Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326)

Ferguson, M. — 2023-05-14

Syphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum (Tp), is resurging globally. Opsonic antibodies (Abs) targeting surface-exposed epitopes of the spirochetes outer membrane proteins (OMPs) are believed to promote macrophage-mediated clearance of the bacterium during infection and are presumed to be key to vaccine development. Tps repertoire of outer membrane proteins includes BamA ({beta}-barrel assembly machinery subunit A/TP0326), the central component of the molecular machine that inserts newly exported OMP precursors into the OM lipid bilayer. BamA is a bipartite protein consisting of an 18-stranded {beta}-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. Antisera directed against BamA ECL4 promote internalization of Tp by rabbit peritoneal macrophages. Herein, we employed a novel two-stage, phage display strategy, termed "Epivolve" (for epitope evolution), to generate five site-directed murine monoclonal Abs (mAbs) targeting a centrally located peptide (S2) of BamA ECL4. Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of BamA ECL4 displayed by a Pyrococcus furiosus thioredoxin (PfTrx) scaffold (PfTrxBamA/ECL4). One mAb containing a unique amino acid sequence in both light and heavy chains showed activity in an opsonophagocytosis assay employing murine bone marrow-derived macrophages. Mice and rabbits hyperimmunized with PfTrxBamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides including S2. In contrast, Abs generated during Tp infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope. Epivolve, which circumvents the natural immune response, can be utilized for the generation of mAbs that target subdominant opsonic epitopes in ECLs of Tp OMPs.

Genome-wide Identification of Zero Nucleotide Recursive Splicing in Drosophila

Michael O Duff — 2014-06-11

Recursive splicing is a process in which large introns are removed in multiple steps by resplicing at ratchet points - 5 splice sites recreated after splicing1. Recursive splicing was first identified in the Drosophila Ultrabithorax (Ubx) gene1 and only three additional Drosophila genes have since been experimentally shown to undergo recursive splicing2,3. Here, we identify 196 zero nucleotide exon ratchet points in 130 introns of 115 Drosophila genes from total RNA sequencing data generated from developmental time points, dissected tissues, and cultured cells. Recursive splicing events were identified by splice junctions that map to annotated 5 splice sites and unannotated intronic 3 splice sites, the presence of the sequence AG/GT at the 3 splice site, and a 5 to 3 gradient of decreasing RNA-Seq read density indicative of co-transcriptional splicing. The sequential nature of recursive splicing was confirmed by identification of lariat introns generated by splicing to and from the ratchet points. We also show that recursive splicing is a constitutive process, and that the sequence and function of ratchet points are evolutionarily conserved. Together these results indicate that recursive splicing is commonly used in Drosophila and provides insight into the mechanisms by which some introns are removed.

Determining Exon Connectivity in Complex mRNAs by Nanopore Sequencing

Mohan Bolisetty — 2015-05-22

Though powerful, short-read high throughput RNA sequencing is limited in its ability to directly measure exon connectivity in mRNAs containing multiple alternative exons located farther apart than the maximum read lengths. Here, we use the Oxford Nanopore MinION sequencer to identify 7,899 full-length isoforms expressed from four Drosophila genes, Dscam1, MRP, Mhc, and Rdl. These results demonstrate that nanopore sequencing can be used to deconvolute individual isoforms and that it has the potential to be an important method for comprehensive transcriptome characterization.

Resources for the comprehensive discovery of functional RNA elements

Balaji Sundararaman — 2015-11-03

Transcriptome-wide maps of RNA binding protein (RBP)-RNA interactions by immunoprecipitation (IP)-based methods such as RNA IP (RIP) and crosslinking and IP (CLIP) are key starting points for evaluating the molecular roles of the thousands of human RBPs. A significant bottleneck to the application of these methods in diverse cell-lines, tissues and developmental stages, is the availability of validated IP-quality antibodies. Using IP followed by immunoblot assays, we have developed a validated repository of 438 commercially available antibodies that interrogate 365 unique RBPs. In parallel, 362 short-hairpin RNA (shRNA) constructs against 276 unique RBPs were also used to confirm specificity of these antibodies. These antibodies can characterize subcellular RBP localization. With the burgeoning interest in the roles of RBPs in cancer, neurobiology and development, these resources are invaluable to the broad scientific community. Detailed information about these resources is publicly available at the ENCODE portal (https://www.encodeproject.org/).nnHighlightsO_LIAntibodies against 365 unique RBPs successfully immunoprecipitate the RBPsnC_LIO_LIShort-hairpin RNAs against 276 unique RBPs confirm the specificity of RBP antibodiesnC_LIO_LIAntibodies characterize subcellular localization of RBPsnC_LIO_LIAntibody and hairpin RNA information are provided at https://www.encodeproject.org/nC_LI

The reported healthy ageing gene expression score: lack of association in two cohorts

Luke C Pilling — 2015-12-09

Sood et al. report a multi-tissue RNA signature "predictive of human health, using only peripheral blood samples". We tested this score in blood in two independent, larger cohorts and found no associations with age or related phenotypes, including muscle strength, interleukin-6 or mortality.

HUMAN LONGEVITY IS INFLUENCED BY MANY GENETIC VARIANTS: EVIDENCE FROM 75,000 UK BIOBANK PARTICIPANTS

Luke C Pilling — 2016-02-01

Variation in human lifespan is 20 to 30% heritable but few genetic variants have been identified. We undertook a Genome Wide Association Study (GWAS) using age at death of parents of middle-aged UK Biobank participants of European decent (n=75,244 with fathers and/or mothers data). Genetic risk scores for 19 phenotypes (n=777 proven variants) were also tested.nnGenotyped variants (n=845,997) explained 10.2% (SD=1.3%) of combined parental longevity. In GWAS, a locus in the nicotine receptor CHRNA3 - previously associated with increased smoking and lung cancer - was associated with paternal age at death, with each protective allele (rs1051730[G]) being associated with 0.03 years later age at fathers death (p=3x10-8). Offspring of longer lived parents had more protective alleles (lower genetic risk scores) for coronary artery disease, systolic blood pressure, body mass index, cholesterol and triglyceride levels, type-1 diabetes, inflammatory bowel disease and Alzheimers disease. In candidate gene analyses, variants in the TOMM40/APOE locus were associated with longevity (including rs429358, p=3x10-5), but FOXO variants were not associated.nnThese results support a multiple protective factors model for achieving longer lifespans in humans, with a prominent role for cardiovascular-related pathways. Several of these genetically influenced risks, including blood pressure and tobacco exposure, are potentially modifiable.

Actomyosin Dynamics Determine the Extension and Retraction of Filopodia on Neuronal Dendrites

Olena O Marchenko — 2016-06-09

Impact StatementIn this study, using a combination of computational and experimental approaches we show that a complex dynamic behavior of dendritic filopodia that is essential for synaptogenesis is explained by an interplay among forces generated by actin retrograde flow, myosin contractility, and substrate adhesion.nnAbstractDendritic filopodia are actin-filled dynamic subcellular structures that sprout on neuronal dendrites during neurogenesis. The exploratory motion of the filopodia is crucial for synaptogenesis but the underlying mechanisms are poorly understood. To study the filopodial motility, we collected and analyzed image data on filopodia in cultured rat hippocampal neurons. We hypothesized that mechanical feedback among the actin retrograde flow, myosin activity and substrate adhesion gives rise to various filopodial behaviors. We have formulated a minimal one-dimensional partial differential equation model that reproduced the range of observed motility. To validate our model, we systematically manipulated experimental correlates of parameters in the model: substrate adhesion strength, actin polymerization rate, myosin contractility and the integrity of the putative microtubule-based barrier at the filopodium base. The model predicts the response of the system to each of these experimental perturbations, supporting the hypothesis that our actomyosin-driven mechanism controls dendritic filopodia dynamics.

Modeling the dynamics of mouse iron body distribution: hepcidin is necessary but not sufficient

Jignesh H. Parmar — 2016-07-08

BackgroundIron is an essential element of most living organisms but is a dangerous substance when poorly liganded in solution. The hormone hepcidin regulates the export of iron from tissues to the plasma contributing to iron homeostasis and also restricting its availability to infectious agents. Disruption of iron regulation in mammals leads to disorders such as anemia and hemochromatosis, and contributes to the etiology of several other diseases such as cancer and neurodegenerative diseases. Here we test the hypothesis that hepcidin alone is able to regulate iron distribution in different dietary regimes in the mouse using a computational model of iron distribution calibrated with radioiron tracer data.nnResultsA model was developed and calibrated to the data from adequate iron diet, which was able to simulate the iron distribution under a low iron diet. However simulation of high iron diet shows considerable deviations from the experimental data. Namely the model predicts more iron in red blood cells and less iron in the liver than what was observed in experiments.nnConclusionsThese results suggest that hepcidin alone is not sufficient to regulate iron homeostasis in high iron conditions and that other factors are important. The model was able to simulate anemia when hepcidin was increased but was unable to simulate hemochromatosis when hepcidin was suppressed, suggesting that in high iron conditions additional regulatory interactions are important.

The Influence of Canalization on the Robustness of Boolean Networks

Claus Kadelka — 2016-07-15

Time- and state-discrete dynamical systems are frequently used to model molecular networks. This paper provides a collection of mathematical and computational tools for the study of robustness in Boolean network models. The focus is on networks governed by k-canalizing functions, a recently introduced class of Boolean functions that contains the well-studied class of nested canalizing functions. The activities and sensitivity of a function quantify the impact of input changes on the function output. This paper generalizes the latter concept to c-sensitivity and provides formulas for the activities and c-sensitivity of general k-canalizing functions as well as canalizing functions with more precisely defined structure. A popular measure for the robustness of a network, the Derrida value, can be expressed as a weighted sum of the c-sensitivities of the governing canalizing functions, and can also be calculated for a stochastic extension of Boolean networks. These findings provide a computationally efficient way to obtain Derrida values of Boolean networks, deterministic or stochastic, that does not involve simulation.

Chaotic propagation of spatial cytoskeletal instability modulates integrity of podocyte foot processes

Cibele V Falkenberg — 2016-07-26

The kidney podocytes function depends on its distinctive morphology. Each podocyte has fingerlike projections, called foot processes, that interdigitate with the processes of neighboring cells to form the glomerular filtration barrier. The integrity of foot process interactions depends on tight spatial control of the dynamics of the underlying actin cytoskeleton, which is regulated by the GTPases, Rac1 and RhoA. To understand how spatially-specific regulation of actin filament dynamics within foot processes controls local morphology, we used a combination of 3-D microscopy and dynamical models. We experimentally determined cell-cell interactions using serial blockface scanning electron microscopy and reconstructed a 3-D spatial representation of a podocyte. We developed a minimal dynamical system for regulation of the actin cytoskeleton; using this 3-D model, we determined how spatial reaction-diffusion dynamics can dysregulate actin bundling, leading to propagation of chaotic foot process effacement. Consistent with experimental observations, our simulations predicted that hyperactive RhoA could destabilize the cytoskeleton. Our simulations showed that deleterious mechanochemical stimuli could lead to local heterogeneity of cytoskeletal dynamics resulting in the emergence of progressive and chaotic loss of foot processes. While global enhancement of Rac1 may result in stronger bundles, the spatial simulations showed that even transient local heterogeneities in polymerization could have dramatic consequences in the stability of multiple foot processes. We conclude that the podocyte morphology optimized for filtration contains intrinsic fragility whereby local imbalances in biochemical and biophysical reactions lead to morphological changes associated with glomerular pathophysiology.

Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins

Belgin Yalçın — 2016-09-29

Axons contain an endoplasmic reticulum (ER) network that is largely smooth and tubular, thought to be continuous with ER throughout the neuron, and distinct in form and function from rough ER; the mechanisms that form this continuous network in axons are not well understood. Mutations affecting proteins of the reticulon or REEP families, which contain intramembrane hairpin domains that can model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). Here, we show that these proteins are required for modeling the axonal ER network in Drosophila. Loss of reticulon or REEP proteins can lead to expansion of ER sheets, and to partial loss of ER from distal motor axons. Ultrastructural analysis reveals an extensive ER network in every axon of peripheral nerves, which is reduced in larvae that lack reticulon and REEP proteins, with defects including larger and fewer tubules, and occasional gaps in the ER network, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of the axonal ER network, suggesting an important role for ER modeling in axon maintenance and function.

Cushing’s Syndrome mutant PKAL205R exhibits altered substrate specificity

Lubner, J. M. — 2016-12-05

The PKAL205R hotspot mutation has been implicated in Cushings Syndrome through hyperactive gain-of-function PKA signaling, however its influence on substrate specificity has not been investigated. Here, we employ the Proteomic Peptide Library (ProPeL) approach to create high-resolution models for PKAWT and PKAL205R substrate specificity. We reveal that the L205R mutation reduces canonical hydrophobic preference at the substrate P+1 position, and increases acidic preference in downstream positions. Using these models, we designed peptide substrates that exhibit altered selectivity for specific PKA variants, and demonstrate the feasibility of selective PKAL205R loss-of-function signaling. Through these results, we suggest that substrate rewiring may contribute to Cushings Syndrome disease etiology, and introduce a powerful new paradigm for investigating mutation-induced kinase substrate rewiring in human disease.

Red Blood Cell Distribution Width: genetic evidence for aging pathways in 116,666 volunteers

Pilling, L. C. — 2017-04-18

Variability in red blood cell volumes (distribution width, RDW) increases with age and is strongly predictive of mortality, incident coronary heart disease and cancer. We investigated inherited genetic variation associated with RDW in 166,666 UK Biobank human volunteers.nnA large proportion RDW is explained by genetic variants (29%), especially in the older group (60+ year olds, 33.8%, <50 year olds, 28.4%). RDW was associated with 194 independent genetic signals; 71 are known for conditions including autoimmune disease, certain cancers, BMI, Alzheimers disease, longevity, age at menopause, bone density, myositis, Parkinsons disease, and age-related macular degeneration. Pathways analysis showed enrichment for telomere maintenance, ribosomal RNA and apoptosis.nnAlthough increased RDW is predictive of cardiovascular outcomes, this was not explained by known CVD or related lipid genetic risks. The predictive value of RDW for a range of negative health outcomes may in part be due to variants influencing fundamental pathways of aging.

Role of Membrane-tension Gated Ca Flux in Cell Mechanosensation

He, L. — 2017-05-04

Eukaryotic cells are sensitive to mechanical forces that they experience from the environment. The process of mechanosensation is complex, and involves elements such as the cytoskeleton and active contraction from myosin motors. Ultimately, mechanosensation is connected to changes in gene expression in the cell, or mechanotransduction. While the involvement of the cytoskeleton in mechanosensation is known, processes upstream to cytoskeletal changes is unclear. In this paper, using a microfluidic device that mechanically compresses live cells, we demonstrate that calcium currents and membrane tension-sensitive ion channels directly signals to the Rho GTPase and myosin contraction. In response to membrane tension changes, cell actively regulates cortical myosin contraction to balance external forces. The process is captured by a mechanochemical model where membrane tension, myosin contraction and the osmotic pressure difference between the cytoplasm and extracellular environment are connected by mechanical force-balance. Finally, to complete the picture of mechanotransduction, we find that the tension-sensitive transcription factor YAP translocates from the nucleus to the cytoplasm in response to mechanical compression.

High Resolution Epigenomic Atlas of Early Human Craniofacial Development

Wilderman, A. — 2017-05-10

Defects in embryonic patterning resulting in craniofacial abnormalities are common birth defects affecting up to 1 in 500 live births worldwide, and are mostly non-syndromic. The regulatory programs that build and shape the craniofacial complex are thought to be controlled by information encoded in the genome between genes and within intronic sequences. Early stages of human craniofacial development have not been interrogated with modern functional genomics techniques, preventing systematic analysis of genetic associations with craniofacial-specific regulatory sequences. Here we describe a comprehensive resource of craniofacial epigenomic annotations and systematic, integrative analysis with a variety of human tissues and cell types. We identified thousands of novel craniofacial enhancers and provide easily accessible genome annotations for craniofacial researchers and clinicians. We demonstrate the utility of our data to find likely causal variants for craniofacial abnormalities and identify a large enhancer cluster that interacts with HOXA genes during craniofacial development.

Ccr4 And Pop2 Control Poly(A) Tail Length In Saccharomyces cerevisiae

Balagopal, V. — 2017-05-19

Messenger RNA degradation is an important aspect of post-transcriptional gene regulation and shortening the poly(A) tail is suggested to be the rate-limiting step in mRNA degradation. In Saccharomyces cerevisiae, the Ccr4-Not complex is the major deadenylase and contains two subunits with exoribonuclease domains, Ccr4 and Pop2. Although the role of Ccr4 and Pop2 in deadenylation has previously been studied using individual reporter mRNAs, their activity has not been studied transcriptome-wide. Here, we describe END-seq, a method to accurately measure poly(A) tail lengths of individual mRNAs transcriptome-wide, and have used this assay to examine the impact of deleting or mutating CCR4 and POP2 on steady state poly(A) tail length. We found that Ccr4 and Pop2 have differential effects on the poly(A) tail lengths of individual mRNAs. Additionally, though Pop2 has previously been reported to have exonuclease activity, mutations that render it catalytically inactive have no effect on steady-state poly(A) tail lengths. Furthermore, mutations that disrupt the interaction between Ccr4 and Pop2 result in longer poly(A) tails. We also observe an inverse correlation between codon optimality and poly(A) tail length - transcripts containing predominantly optimal codons display fewer changes in poly(A) tail length upon deletion of Ccr4 or Pop2 than those containing less optimal codons. Together, these results indicate that Pop2 modulates poly(A) tail length, at least partially, via its association with Ccr4 and that Pop2 improves the function of Ccr4 in regulating poly(A) tail length. These data provide important insights into poly(A) tail length dynamics in yeast and demonstrate that END-seq is an efficient and accurate method to study poly(A) tail length.

Compartmental and spatial rule-based modeling with Virtual cell (VCell)

Blinov, M. L. — 2017-06-05

In rule-based modeling, molecular interactions are systematically specified in the form of reaction rules that serve as generators of reactions. This provides a way to account for all the potential molecular complexes and interactions among multivalent or multistate molecules. Recently, we introduced rule- based modeling into the Virtual Cell (VCell) modeling framework, permitting graphical specification of rules and merger of networks generated automatically (using the BioNetGen modeling engine) with hand-specified reaction networks. VCell provides a number of ordinary differential equation (ODE) and stochastic numerical solvers for single-compartment simulations of the kinetic systems derived from these networks, and agent-based network-free simulation of the rules. In this work, compartmental and spatial modeling of rule-based models has been implemented within VCell. To enable rule-based deterministic and stochastic spatial simulations and network-free agent-based compartmental simulations, the BioNetGen and NFSim engines were each modified to support compartments. In the new rule-based formalism, every reactant and product pattern and every reaction rule are assigned locations. We also introduce the novel rule-based concept of molecular anchors. This assures that any species that has a molecule anchored to a predefined compartment will remain in this compartment. Importantly, in addition to formulation of compartmental models, this now permits VCell users to seamlessly connect reaction networks derived from rules to explicit geometries to automatically generate a system of reaction-diffusion equations. These may then be simulated using either the VCell partial differential equations (PDE) deterministic solvers or the Smoldyn stochastic simulator.

Addressing current challenges in cancer immunotherapy with mathematical and computational modeling

Konstorum, A. — 2017-06-09

The goal of cancer immunotherapy is to boost a patients immune response to a tumor. Yet, the design of an effective immunotherapy is complicated by various factors, including a potentially immunosuppressive tumor microenvironment, immune-modulating effects of conventional treatments, and therapy-related toxicities. These complexities can be incorporated into mathematical and computational models of cancer immunotherapy that can then be used to aid in rational therapy design. In this review, we survey modeling approaches under the umbrella of the major challenges facing immunotherapy development, which encompass tumor classification, optimal treatment scheduling, and combination therapy design. Although overlapping, each challenge has presented unique opportunities for modelers to make contributions using analytical and numerical analysis of model outcomes, as well as optimization algorithms. We discuss several examples of models that have grown in complexity as more biological information has become available, showcasing how model development is a dynamic process interlinked with the rapid advances in tumor-immune biology. We conclude the review with recommendations for modelers both with respect to methodology and biological direction that might help keep modelers at the forefront of cancer immunotherapy development.

Langevin Dynamics Simulation of AKAP-PKA Complex: Re-Envisioning the Local Concentration Mechanism for Directing PKA Phosphorylation

Rigatti, M. — 2017-07-22

The second messenger cAMP and its effector cAMP-dependent protein kinase A (PKA) constitute a ubiquitous cell signaling system. In its inactive state PKA is composed of two regulatory subunits that dimerize, and two catalytic subunits that are inhibited by the regulatory subunits. Activation of the catalytic subunits occurs upon binding of two molecules of cAMP to each regulatory subunit. Although many receptor types existing within the same cell may use this signaling system, compartmentation of signaling is thought to occur due to A-Kinase Anchoring Proteins (AKAPs), which act to co-localize PKA with specific substrates. However, the molecular mechanism allowing AKAPs to direct PKA phosphorylation to a particular substrate remained elusive, as prior evidence suggested that the catalytic subunit, which is highly diffusible, is released after cAMP binding to the regulatory subunit. Recent evidence from Smith et al. suggests that in the cell, the catalytic subunit may in fact not be released from the AKAP complex [1, 2]. They further demonstrated that alterations in the structure of the PKA regulatory subunit tether affect substrate phosphorylation. We use a novel computational software based on Langevin dynamics, SpringSaLaD, to simulate the AKAP-PKA complex in order to determine a molecular mechanism for the changes in phosphorylation seen with alteration in tether length and flexibility, and to demonstrate whether or not AKAPs can effectively direct PKA phosphorylation to a particular substrate upon release of the catalytic subunit from the complex. We find that short and flexible tethers contribute to a decrease in the average characteristic time of binding, allowing the catalytic subunit to spend more time in a bound state with the substrate, which yields faster characteristic times of phosphorylation. We further demonstrate that release of the catalytic subunit from the AKAP complex abrogates the effect of tethering, with characteristic times of phosphorylation similar to non-AKAP bound PKA. The data demonstrates that AKAPs likely do not release the catalytic subunit in directing PKA phosphorylation to AKAP bound substrates. In combination with the changes in characteristic time of phosphorylation which are driven by tether structure, this work indicates that the purpose of AKAPs may be to increase the efficiency of phosphorylation of particular AKAP substrates.

A Large-Scale Binding and Functional Map of Human RNA Binding Proteins

Van Nostrand, E. L. — 2017-08-23

Genomes encompass all the information necessary to specify the development and function of an organism. In addition to genes, genomes also contain a myriad of functional elements that control various steps in gene expression. A major class of these elements function only when transcribed into RNA as they serve as the binding sites for RNA binding proteins (RBPs), which act to control post-transcriptional processes including splicing, cleavage and polyadenylation, RNA editing, RNA localization, stability, and translation. Despite the importance of these functional RNA elements encoded in the genome, they have been much less studied than genes and DNA elements. Here, we describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. These data expand the catalog of functional elements encoded in the human genome by addition of a large set of elements that function at the RNA level through interaction with RBPs.nnHighlightsO_LI223 eCLIP datasets for 150 RBPs reveal a wide variety of in vivo RNA target classes.nC_LIO_LI472 knockdown/RNA-seq profiles of 263 RBPs reveal factor-responsive targets and integration with eCLIP indicates RNA expression and splicing regulatory patterns.nC_LIO_LI78 RNA Bind-N-Seq profiles of in vitro binding motifs reveal links between in vitro and in vivo binding and indicate that eCLIP peaks that contain in vitro motifs are more strongly associated with regulation.nC_LIO_LI274 maps of RBP subcellular localization by immunofluorescence indicate widespread organelle-specific RNA processing regulation.nC_LIO_LI63 ChIP-seq profiles of DNA association suggest broad interconnectivity between chromatin association and RNA processing.nC_LI

High-Throughput Identification of Genetic Variation Impact on pre-mRNA Splicing Efficiency

Adamson, S. I. — 2017-09-20

AbstractUnderstanding the functional impact of genomic variants is a major goal of modern genetics and personalized medicine. Although many synonymous and non-coding variants act through altering the efficiency of pre-mRNA splicing, it is difficult to predict how these variants impact pre-mRNA splicing. Here, we describe a massively parallel approach we used to test the impact of 2,059 human genetic variants spanning 110 alternative exons on pre-mRNA splicing. This method yields data that reinforces known mechanisms of pre-mRNA splicing, can rapidly identify genomic variants that impact pre-mRNA splicing, and will be useful for increasing our understanding of genome function.

Dephosphorylation is the Mechanism of Fibroblast Growth Factor Inhibition of Guanylyl Cyclase-B

Robinson, J. W. — 2017-09-20

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations of guanylyl cyclase-B (GC-B, also called NPRB or NPR2) cause dwarfism. FGF exposure inhibits GC-B activity in a chondrocyte cell line, but the mechanism of the inactivation is not known. Here, we report that FGF exposure causes dephosphorylation of GC-B in rat chondrosarcoma cells, which correlates with a rapid, potent and reversible inhibition of C-type natriuretic peptide-dependent activation of GC-B. Cells expressing a phosphomimetic mutant of GC-B that cannot be inactivated by dephosphorylation because it contains glutamate substitutions for all known phosphorylation sites showed no decrease in GC-B activity in response to FGF. We conclude that FGF rapidly inactivates GC-B by a reversible dephosphorylation mechanism, which may contribute to the signaling network by which activated FGFR3 causes dwarfism.nnHighlightsO_LIGuanylyl Cyclase-B is expressed in rat chondrosarcoma cellsnC_LIO_LIFGF2 induces a rapid, potent, and reversible inhibition of GC-BnC_LIO_LIFGF2 treatment causes GC-B dephosphorylationnC_LIO_LIFGF2 does not inhibit a dephosphorylation-resistant form of GC-BnC_LIO_LIDephosphorylation is the mechanism of FGF2-dependent inhibition of GC-BnC_LInnAbbreviations

Dephosphorylation of the NPR2 guanylyl cyclase contributes to inhibition of bone growth by fibroblast growth factor

Shuhaibar, L. C. — 2017-09-25

Activating mutations in fibroblast growth factor (FGF) receptor 3 and inactivating mutations in the NPR2 guanylyl cyclase cause similar forms of dwarfism, but how these two signaling systems interact to regulate bone growth is poorly understood. Here, by use of a mouse model in which NPR2 cannot be dephosphorylated, we show that bone elongation is opposed when NPR2 is dephosphorylated and thus produces less cyclic GMP. By developing an in vivo imaging system to measure cyclic GMP levels in intact tibia, we show that FGF-induced dephosphorylation of NPR2 decreases its guanylyl cyclase activity in growth plate chondrocytes in living bone. Thus FGF signaling lowers cyclic GMP in the growth plate, which counteracts bone elongation. These results define a new component of the signaling network by which activating mutations in the FGF receptor inhibit bone growth.

Core-2 O-glycans are required for Galectin-3 interaction with the osteoarthritis related protein lubricin

Flowers, S. A. — 2019-12-21

Synovial fluid lubricin (proteoglycan 4) is a mucin-type O-linked glycosylated (60% of the mass) biological lubricant involved in osteoarthritis (OA) development. Lubricin has been reported to be cross-linked by synovial galectin-3 on the lubricating articular surface. Here, we confirm that binding to galectin-3 depended on core-2 O-linked glycans, where surface plasmon resonance of a recombinant lubricin (rhPRG4) devoid of core-2 structures lacked binding capacity to recombinant galectin-3. Both galectin-3 levels and interactions with synovial lubricin were found to be decreased in late-stage OA patients coinciding with an increase of truncated and less sialylated core 1 O-glycans. These data suggest a defect cross-linking of surface active molecules in OA and provides novel insights into OA molecular pathology.

Sequence, Structure and Context Preferences of Human RNA Binding Proteins

Dominguez, D. — 2017-10-12

Production of functional cellular RNAs involves multiple processing and regulatory steps principally mediated by RNA binding proteins (RBPs). Here we present the affinity landscapes of 78 human RBPs using an unbiased assay that determines the sequence, structure, and context preferences of an RBP in vitro from deep sequencing of bound RNAs. Analyses of these data revealed several interesting patterns, including unexpectedly low diversity of RNA motifs, implying frequent convergent evolution of binding specificity toward a relatively small set of RNA motifs, many with low compositional complexity. Offsetting this trend, we observed extensive preferences for contextual features outside of core RNA motifs, including spaced "bipartite" motifs, biased flanking nucleotide context, and bias away from or towards RNA structure. These contextual features are likely to enable targeting of distinct subsets of transcripts by different RBPs that recognize the same core motif. Our results enable construction of "RNA maps" of RBP activity without requiring crosslinking-based assays, and provide unprecedented depth of information on the interaction of RBPs with RNA.

Why most transporter mutations that cause antibiotic resistance are to efflux pumps rather than to import transporters

Mendes, P. — 2020-01-17

Genotypic microbial resistance to antibiotics with intracellular targets commonly arises from mutations that increase the activities of transporters (pumps) that cause the efflux of intracellular antibiotics. A priori it is not obvious why this is so much more common than are mutations that simply inhibit the activity of uptake transporters for the antibiotics. We analyse quantitatively a mathematical model consisting of one generic equilibrative transporter and one generic concentrative uptake transporter (representing any number of each), together with one generic efflux transporter. The initial conditions are designed to give an internal concentration of the antibiotic that is three times the minimum inhibitory concentration (MIC). The effect of varying the activity of each transporter type 100-fold is dramatically asymmetric, in that lowering the activities of individual uptake transporters has comparatively little effect on internal concentrations of the antibiotic. By contrast, increasing the activity of the efflux transporter lowers the internal antibiotic concentration to levels far below the MIC. Essentially, these phenomena occur because inhibiting individual influx transporters allows others to take up the slack, whereas increasing the activity of the generic efflux transporter cannot easily be compensated. The findings imply strongly that inhibiting efflux transporters is a much better approach for fighting antimicrobial resistance than is stimulating import transporters. This has obvious implications for the development of strategies to combat the development of microbial resistance to antibiotics and possibly also cancer therapeutics in human.

Complex networks of miRNA-transcription factors mediate gene dosage compensation in aneuploid cancer

Acon, M. S. — 2020-02-01

Cancer complexity is consequence of enormous genomic instability leading to aneuploidy, a hallmark of most cancers. We hypothesize that dosage compensation of critical genes could arise from systems-level properties of complex networks of microRNAs (miRNA) and transcription factors (TF) as a way for cancer cells to withstand the negative effects of aneuploidy. We studied gene dosage compensation at the transcriptional level on data of the NCI-60 cancer cell line panel with the aid of computational models to identify candidate genes with low tolerance to variation in gene expression despite high variation in copy numbers. We identified a network of TF and miRNAs validated interactions with those genes to construct a mathematical model where the property of dosage compensation emerged for MYC and STAT3. Compensation was mediated by feedback and feed-forward motifs with 4 miRNAs and was dependent on the kinetic parameters of these TF-miRNA interactions, indicating that network analysis was not enough to identify this emergent property. The inhibition of miRNAs compensating MYC suggest a therapeutic potential of targeting gene dosage compensation against aneuploid cancer.

Cellular heterogeneity of the LH receptor and its significance for cyclic GMP signaling in mouse preovulatory follicles

Baena, V. — 2020-02-07

Meiotic arrest and resumption in mammalian oocytes are regulated by two opposing signaling proteins in the cells of the surrounding follicle: the guanylyl cyclase NPR2, and the luteinizing hormone receptor (LHR). NPR2 maintains a meiosis-inhibitory level of cyclic GMP (cGMP) until LHR signaling causes dephosphorylation of NPR2, reducing NPR2 activity, lowering cGMP to a level that releases meiotic arrest. However, the signaling pathway between LHR activation and NPR2 dephosphorylation remains incompletely understood, due in part to imprecise information about the cellular localization of these two proteins. To investigate their localization, we generated mouse lines in which HA epitope tags were added to the endogenous LHR and NPR2 proteins, and used immunofluorescence and immunogold microscopy to localize these proteins with high resolution. The results showed that the LHR protein is absent from the cumulus cells and inner mural granulosa cells, and is present in only 13-48% of the outer mural granulosa cells. In contrast, NPR2 is present throughout the follicle, and is more concentrated in the cumulus cells. Less than 20% of the NPR2 is in the same cells that express the LHR. These results suggest that to account for the LH-induced inactivation of NPR2, LHR-expressing cells send a signal that inactivates NPR2 in neighboring cells that do not express the LHR. An inhibitor of gap junction permeability attenuates the LH-induced cGMP decrease in the outer mural granulosa cells, consistent with this mechanism contributing to how NPR2 is inactivated in cells that do not express the LHR.

Macrophage scavenger receptor 1 controls Chikungunya virus infection through autophagy

Yang, L. — 2020-03-02

Macrophage scavenger receptor 1 (MSR1) mediates the endocytosis of modified low-density lipoproteins and plays an important antiviral role. However, the molecular mechanism underlying MSR1 antiviral actions remains elusive. Herein, we report that MSR1 activates autophagy to restrict infection of Chikungunya virus (CHIKV), an arthritogenic alphavirus that causes acute and chronic crippling arthralgia. Msr1 expression was rapidly upregulated after CHIKV infection in mice. Msr1 knockout mice had elevated viral loads and increased susceptibility to CHIKV arthritis along with a normal type I IFN response. Induction of LC3 lipidation by CHIKV, a marker of autophagy, was reduced in Msr1-/- cells. Mechanistically, MSR1 interacted with ATG12 through its cytoplasmic tail and this interaction was enhanced by CHIKV nsP1 protein. MSR1 repressed CHIKV replication through ATG5-ATG12-ATG16L1 and this was dependent on the FIP200-and-WIPI2-binding domain, but not the WD40 domain of ATG16L1. Our results elucidate an antiviral role for MSR1 involving the autophagic function of ATG5-ATG12-ATG16L1.

A Single Membrane Protein Required for Atrial Secretory Granule Formation

Back, N. — 2020-03-09

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is Peptidylglycine -Amidating Monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (PamMyh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult PamMyh6-cKO/cKO atria revealed a 20-fold drop in the volume fraction of secretory granules and a decrease in peripherally localized Golgi complexes. When primary cultures of Pam0-Cre-cKO/cKO atrial myocytes (PAM floxed, no Cre recombinase) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in proANP levels. Expression of exogenous PAM in PamMyh6-cKO/cKO atrial myocytes produced a dose-dependent increase in proANP content. Strikingly, rescue of proANP content did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact and its basal secretion is stimulated by drugs that inhibit Golgi-localized Arf activators. Increased basal secretion of proANP was a major contributor to its reduced levels in PamMyh6-cKO/cKO myocytes; the inability of these drugs to inhibit basal proANP secretion by PamMyh6-cKO/cKO myocytes revealed a role for COPI-mediated recycling of PAM to the endoplasmic reticulum. Analysis of atrial coated vesicles and the ability PAM to make fluorescently-tagged proANP accumulate in the cis-Golgi region of cells lacking secretory granules revealed a non-catalytic role for PAM in soluble cargo trafficking early in the secretory pathway. SignificanceTransmission electron microscopy of atrial cardiomyocytes revealed dense granules resembling those in endocrine cells and neurons, leading to the discovery of the natriuretic peptides stored in these granules. Subsequent studies revealed features unique to atrial granules, including high level expression of Peptidylglycine -Amidating Monooxygenase (PAM), an enzyme required for the synthesis of many neuropeptides, but not for the synthesis of natriuretic peptides. The discovery that atrial myocytes lacking PAM are unable to produce granules and that PAM lacking its monooxygenase activity can rescue granule formation provides new information about the proANP secretory pathway. A better understanding of the unique features of atrial cell biology should provide insight into atrial fibrillation, the most common cardiac arrhythmia, atrial amyloidosis and heart failure.

Antibiotic hypersensitivity signatures identify targets for attack in the Acinetobacter baumannii cell envelope

Geisinger, E. — 2020-03-12

Acinetobacter baumannii is an opportunistic pathogen that is a critical, high-priority target for new antibiotic development. Clearing of A. baumannii requires relatively high doses of antibiotics across the spectrum, primarily due to its protective cell envelope. Many of the proteins that support envelope integrity and modulate drug action are uncharacterized, largely because there is an absence of orthologs for several proteins that perform essential envelope-associated processes, impeding progress on this front. To identify targets that can synergize with current antibiotics, we performed an exhaustive analysis of A. baumannii mutants causing hypersensitivity to a multitude of antibiotic treatments. By examining mutants with antibiotic hypersensitivity profiles that parallel mutations in proteins of known function, we show that the function of poorly annotated proteins can be predicted and used to identify candidate missing link proteins in essential A. baumannii processes. Using this strategy, we uncovered multiple uncharacterized proteins with critical roles in cell division or cell elongation, and revealed that a predicted cell wall D,D-endopeptidase has an unappreciated function in lipooligosaccharide synthesis. Moreover, we provide a genetic strategy that uses hypersensitivity signatures to predict drug synergies, allowing the identification of {beta}-lactams that work cooperatively based on the cell wall assembly machineries that they preferentially target. These data reveal multiple pathways critical for envelope growth in A. baumannii that can be targeted in combination strategies for attacking the pathogen.

Structural and developmental principles of neuropil assembly in C. elegans

Moyle, M. W. — 2020-03-15

Neuropil is a fundamental form of tissue organization within brains1. In neuropils, densely packed neurons synaptically interconnect into precise circuit architecture2,3, yet the structural and developmental principles governing nanoscale precision in bundled neuropil assembly remain largely unknown4-6. Here we use diffusion condensation, a coarse-graining clustering algorithm7, to identify nested circuit structures within the C. elegans cerebral neuropil (called the nerve ring). We determine that the nerve ring neuropil is organized into four tightly bundled strata composed of related behavioral circuits. We demonstrate that the stratified architecture of the neuropil is a geometrical representation of the functional segregation of sensory information and motor outputs, with specific sensory organs and muscle quadrants mapping onto particular neuropil strata. We identify groups of neurons with unique morphologies that integrate information across strata and that create a sophisticated honeycomb-shaped scaffold that encases the strata within the nerve ring. We resolve the developmental sequence leading to stratified neuropil organization through the integration of lineaging and cell tracking algorithms with high resolution light-sheet microscopy, and reveal principles of cell position, migration and hierarchical outgrowth that guide neuropil organization. Our results uncover conserved design principles underlying nerve ring neuropil architecture and function, and a pioneer neuron-based, temporal progression of outgrowth that guides the hierarchical development of the layered neuropil. Our findings provide a blueprint for using structural and developmental approaches to systematically understand neuropil organization within brains.

Identifying dynamic protein and RNA proximity interaction networks of actinin reveals RNA-binding and metabolic regulatory mechanisms

Ladha, F. A. — 2020-03-19

RationaleActinins are actin cross-linkers that are ubiquitously expressed and harbor mutations in heritable diseases. The predominant cardiac actinin is encoded by ACTN2, which is a core structural component of the sarcomere Z-disk. ACTN2 is required for sarcomere function through complex interactions with proteins involved in sarcomere assembly, cell signaling and transcriptional regulation. However, there remain critical gaps in our knowledge of the complete and dynamic cardiac actinin interactome, which could reveal new insights into sarcomere biology. ObjectiveWe sought to examine the cardiac actinin interactome through sarcomere assembly in human cardiomyocytes. Methods and ResultsWe utilized CRISPR/Cas9, induced pluripotent stem cell technology and BioID to reveal cardiac actinin protein interactions in human cardiomyocytes. We identified 324 cardiac actinin proximity partners, analyzed networks, and studied interactome changes associated with sarcomere assembly. We focused additional studies on unexpected actinin interactions with effectors with RNA-binding functions. Using RNA immunoprecipitation followed by sequencing, we determined that RNA-binding partners uncovered by actinin BioID were bound to gene transcripts with electron transport chain and mitochondrial biogenesis functions. Mammalian two-hybrid studies established that IGF2BP2, an RNA-binding protein associated with type 2 diabetes, directly interacted with the rod domain of actinin through its K Homology domain. IGF2BP2 was necessary for electron transport chain transcript localization to vicinal RNA-binding proteins, mitochondrial mass and oxidative metabolism. IGF2BP2 knockdown also impaired sarcomere function in a cardiac microtissue assay. ConclusionsThis study expands our functional knowledge of cardiac actinin, uncovers new sarcomere interaction partners including those regulated by sarcomere assembly, and reveals sarcomere crosstalk with RNA-binding proteins including IGF2BP2 that are important for metabolic functions.

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus

Ding, X. — 2020-03-21

A recent outbreak of novel coronavirus (SARS-CoV-2), the causative agent of COVID-19, has spread rapidly all over the world. Human immunodeficiency virus (HIV) is another deadly virus and causes acquired immunodeficiency syndrome (AIDS). Rapid and early detection of these viruses will facilitate early intervention and reduce disease transmission risk. Here, we present an All-In-One Dual CRISPR-Cas12a (termed "AIOD-CRISPR") assay method for simple, rapid, ultrasensitive, one-pot, and visual detection of coronavirus SARS-CoV-2 and HIV virus. In our AIOD CRISPR assay, a pair of crRNAs was introduced to initiate dual CRISPR-Cas12a detection and improve detection sensitivity. The AIOD-CRISPR assay system was successfully utilized to detect nucleic acids (DNA and RNA) of SARS-CoV-2 and HIV with a sensitivity of few copies. Also, it was evaluated by detecting HIV-1 RNA extracted from human plasma samples, achieving a comparable sensitivity with real-time RT-PCR method. Thus, our method has a great potential for developing next-generation point-of-care molecular diagnostics.

Dynamic aqueous multiphase reaction system for simple, sensitive and quantitative one-pot CRISPR-Cas12a based molecular diagnosis

Yin, K. — 2020-03-24

Recently, CRISPR-Cas technology has opened a new era of nucleic acid-based molecular diagnostics. However, current CRISPR-Cas-based nucleic acid biosensing has largely a lack of the quantitative detection ability and typically requires separate manual operations. Herein, we reported a dynamic aqueous multiphase reaction (DAMR) system for simple, sensitive and quantitative one-pot CRISPR-Cas12a based molecular diagnosis by taking advantage of density difference of sucrose concentration. In the DAMR system, recombinase polymerase amplification (RPA) and CRISPR-Cas12a derived fluorescent detection occurred in spatially separated but connected aqueous phases. Our DAMR system was utilized to quantitatively detect human papillomavirus (HPV) 16 and 18 DNAs with sensitivities of 10 and 100 copies within less than one hour. Multiplex detection of HPV16/18 in clinical human swab samples were successfully achieved in the DAMR system using 3D-printed microfluidic device. Furthermore, we demonstrated that target DNA in real human plasma samples can be directly amplified and detected in the DAMR system without complicated sample pre-treatment. As demonstrated, the DAMR system has shown great potential for development of next-generation point-of-care molecular diagnostics.

Abundance and localization of human UBE3A protein isoforms

Sirois, C. L. — 2020-03-31

Loss of UBE3A expression, a gene regulated by genomic imprinting, causes Angelman Syndrome (AS), a rare neurodevelopmental disorder. The UBE3A gene encodes an E3 ubiquitin ligase with three known protein isoforms in humans. Studies in mouse suggest that the human isoforms may have differences in localization and neuronal function. A recent case study reported mild AS phenotypes in individuals lacking one specific isoform. Here we have used CRISPR/Cas9 to generate isogenic human embryonic stem cells (hESCs) that lack the individual protein isoforms. We demonstrate that isoform 1 accounts for the majority of UBE3A protein in hESCs and neurons. We also show that UBE3A predominantly localizes to the cytoplasm in both wild type and isoform-null cells. Finally, we show that neurons lacking isoform 1 display a less severe electrophysiological AS phenotype.

Testosterone use in female mice does not impair fertilizability of eggs: Implications for the fertility care of transgender males

Bartels, C. B. — 2020-04-11

STUDY QUESTIONDoes testosterone use in females affect reproductive potential, particularly with regard to the production of fertilizable gametes? SUMMARY ANSWERTestosterone cypionate injections given to post-pubertal female mice caused virilization and ovaries were smaller than control ovaries, but ovaries were still responsive to hormonal stimulation and produced fertilizable eggs when superovulated. WHAT IS KNOWN ALREADYStudies to examine the effects of testosterone on reproductive potential in transgender males are lacking. Recently, a model was developed that simulates many aspects of testosterone use in transgender males in order to look at reproductive effects of testosterone in female mice. This study found masculinizing effects on the mice but did not find significant deficits on the number of ovarian follicles; however, effects of testosterone use on ovarian stimulation and fertilizability of oocytes were not investigated. STUDY DESIGN, SIZE, DURATIONA total of 66, 6-week-old Hsd:NSA(CF-1) female mice and 6 Hsd:ICR (CD-1) mice were used for this study. Mice were injected subcutaneously with 400 g testosterone cypionate or sesame oil once a week for 6 weeks and were either sacrificed a week after the 6th injection (active exposure group), or were sacrificed 6-7 weeks after the final testosterone injection (washout group). PARTICIPANTS/MATERIALS, SETTING, METHODSBoth active exposure and washout groups were further subdivided into 3 groups: unstimulated, eCG-stimulated, or eCG/hCG-stimulated. eCG-stimulated mice were sacrificed 44-48 hrs after eCG injection. eCG/hCG-stimulated mice were injected with eCG, followed 48 hrs later with hCG. Mice were sacrificed [~]13-18 hrs after the hCG injection. Data collected included daily vaginal cytology, terminal hormone levels and ovary weights, ovarian histology, number of oocytes/eggs collected in each group, and cleavage to the 2-cell stage following in vitro fertilization. MAIN RESULTS AND THE ROLE OF CHANCETestosterone cypionate-treated mice had testosterone levels elevated to the level of male mice and ceased cycling. Ovaries were significantly smaller in testosterone-treated mice, but they contained normal cohorts of follicles and responded to gonadotropin stimulation by ovulating similar numbers of eggs that fertilized and cleaved in vitro. LIMITATIONS, REASONS FOR CAUTIONOur model treated female mice for only 6 weeks, whereas many transgender men use testosterone for many years before considering biological children. Importantly, a mouse system may not perfectly simulate human reproductive physiology. WIDER IMPLICATIONS OF THE FINDINGSThe current standard of care for transgender men who desire biological children is to cease testosterone therapy prior to ovarian stimulation, but the necessity for stopping testosterone is not known. Our model demonstrates that it is possible for testosterone-suppressed ovaries to respond to gonadotropic stimulation by producing and ovulating fertilizable eggs, thereby obviating the need for testosterone cessation prior to ovarian stimulation. In time, these results may provide insights for future clinical trials of fertility treatment options for transgender men.

Analysis of Treponema pallidum strains from China using improved methods for whole-genome sequencing from primary syphilis chancres

Chen, W. — 2020-04-15

Whole-genome sequencing (WGS) of Treponema pallidum subsp. pallidum (TPA) has been constrained by the lack of in vitro cultivation methods for isolating spirochetes from patient samples. We built upon recently developed enrichment methods to sequence TPA directly from primary syphilis chancre swabs collected in Guangzhou, China. By combining parallel, pooled whole-genome amplification (ppWGA) with hybrid selection, we generated high quality genomes from four of eight chancre-swab samples and two of two rabbit-passaged isolates, all subjected to challenging storage conditions. This approach enabled the first WGS of Chinese samples without rabbit passage and provided insights into TPA genetic diversity in China.

CD13 is a Critical Regulator of Cell-cell Fusion in Osteoclastogenesis

Ghosh, M. — 2020-04-25

In vertebrates, bone formation is dynamically controlled by the activity of two specialized cell types: the bone-generating osteoblasts and bone-degrading osteoclasts. Osteoblasts produce the soluble receptor activator of NFκB ligand (RANKL) that binds to its receptor RANK on the surface of osteoclast precursor cells to promote osteoclastogenesis, a process that involves cell-cell fusion and assembly of molecular machinery to ultimately degrade the bone. CD13 is a transmembrane aminopeptidase that is highly expressed in cells of myeloid lineage has been shown to regulate dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. In the present study, we show that CD13-deficient mice display a normal distribution of osteoclast progenitor populations in the bone marrow, but present a low bone density phenotype. Further, the endosteal bone formation rate is similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Loss of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells with remarkably high numbers of nuclei with a concomitant increase in bone resorption activity. Similarly, we also observed increased formation of multinucleated giant cells (MGC) in CD13KO bone marrow progenitor cells stimulated with IL-4 and IL-13, suggesting that CD13 may regulate cell-cell fusion events via a common pathway, independent of RANKL signaling. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP are normally downregulated as fusion progresses in fusion-competent mononucleated progenitor cells, in the absence of CD13 they are uniformly sustained at high levels, even in mature multi-nucleated osteoclasts. Taken together, we conclude that CD13 may regulate cell-cell fusion by controlling expression and localization of key fusion proteins that are critical for both osteoclast and MGC fusion.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Developmental Patterning of Irritability Enhances Prediction of Psychopathology in Pre-adolescence: Improving RDoC with Developmental Science

Damme, K. S. F. — 2020-04-30

Research has demonstrated the transdiagnostic importance of irritability in psychopathology pathways but the contribution of developmentally-unfolding patterns has only recently been explored. To address this question, irritability patterns of 110 youth from a large and diverse early childhood cohort were assessed at preschool age and at school age ([~]2.5 years later) with a dimensional irritability scale designed to capture the normal:abnormal spectrum. Participants then returned at Pre-adolescence ([~]6 years later) for an assessment with a structured clinical interview (internalizing/externalizing symptoms) and a magnetic resonance imaging scan. When only preschool age irritability was considered, this was a transdiagnostic predictor of internalizing and externalizing symptoms. However, a model including both preschool and school age irritability provided a more nuanced picture. A high preschool and decreasing school age profile of irritability predicted elevated pre-adolescence internalizing symptoms, potentially reflecting emerging coping/internalizing behavior in pre-adolescence. In contrast, a stable irritability profile across these timepoints predicted increased pre-adolescence externalizing symptoms. Further, preschool irritability (a period of rapid growth) did not predict pre-adolescent gray matter volume abnormality, an indicator of transdiagnostic clinical risk. However, irritability at school age (when gray matter volume growth is largely finished) demonstrated an interactive effect among regions; increased school age irritability predicted reduced volume in pre-adolescence emotional regions (e.g., amygdala, medial orbitofrontal cortex) and increased volume in other regions (e.g., cerebellum). Expanding the impact of RDoCs approach yielding transdiagnostic phenotypes and multiple units of analysis, a developmentally informed approach provides critical new insights into the complex unfolding of mechanisms underlying emerging psychopathology.

STING deficiency-associated aberrant CXCL10 expression contributes to pathogenesis of arthritogenic alphaviruses

Lin, T. — 2020-05-15

Arthritogenic alphaviruses such as Chikungunya virus and Onyong nyong virus cause acute and chronic crippling arthralgia associated with inflammatory immune responses. However, the physiological functions of individual immune signaling pathways in the pathogenesis of alphaviral arthritis remain poorly understood. Here we report that a deficiency in the stimulator-of-interferon-genes (STING) led to enhanced viral loads, exacerbated inflammation and selectively elevated expression of CXCL10, a chemoattractant for monocytes/macrophages/T cells, in mouse feet. Cxcl10-/- mice had the same viremia as wild-type animals, but fewer immune infiltrates and lower viral loads in footpads at the peak of arthritic disease (6-8 days post infection). Macrophages constituted the largest immune cell population in footpads following infection, which were significantly reduced in Cxcl10-/- mice. The viral RNA loads in neutrophils and macrophages were reduced in Cxcl10-/- compared to wild-type mice. In summary, our results demonstrate that STING signaling represses, while CXCL10 signaling promotes, pathogenesis of alphaviral disease.

Cellular taxonomy and spatial organization of the ventral posterior hypothalamus reveals neuroanatomical parcellation of the mammillary bodies

Mickelsen, L. E. — 2020-05-15

The ventral posterior hypothalamus (VPH) is an anatomically complex brain region implicated in arousal, reproduction, energy balance and memory processing. However, neuronal cell type diversity within the VPH is poorly understood, an impediment to deconstructing the roles of distinct VPH circuits in physiology and behavior. To address this question, we employed a droplet-based single cell RNA sequencing (scRNA-seq) approach to systematically classify molecularly distinct cell types in the mouse VPH. Analysis of >16,000 single cells revealed 20 neuronal and 18 non-neuronal cell populations, defined by suites of discriminatory markers. We validated differentially expressed genes in a selection of neuronal populations through fluorescence in situ hybridization (FISH). Focusing on the mammillary bodies (MB), we discovered transcriptionally-distinct clusters that exhibit a surprising degree of segregation within neuroanatomical subdivisions of the MB, while genetically-defined MB cell types project topographically to the anterior thalamus. This single cell transcriptomic atlas of cell types in the VPH provides a detailed resource for interrogating the circuit-level mechanisms underlying the diverse functions of VPH circuits in health and disease.

Contiguous Erosion of the Inactive X in Human Pluripotency Concludes With Global DNA Hypomethylation

Bansal, P. — 2020-05-22

Female human pluripotent stem cells (hPSCs) are prone to undergoing X chromosome erosion (XCE), a progressive loss of key epigenetic features on the inactive X that initiates with repression of XIST, the long non-coding RNA required for X inactivation. As a result, previously silenced genes on the eroding X (Xe) reactivate, some of which are thought to provide selective advantages. To-date, the sporadic and progressive nature of XCE has largely obscured its scale, dynamics, and key transition events. To address this knowledge gap, we performed an integrated analysis of DNA methylation (DNAme), chromatin accessibility, and gene expression across hundreds of hPSC samples. Differential methylation across the Xe enables ordering female hPSCs across a trajectory of XCE from initiation to terminal stages. Our results identify a crucial cis-regulatory element for XIST expression, trace contiguously growing domains of reactivation to a few euchromatic origins on the Xi, and indicate that the late-stage Xe impairs DNAme genome-wide. Surprisingly, from this altered epigenetic landscape emerge select features of naive pluripotency, suggesting its link to X chromosome dosage may be partially conserved in human embryonic development.

Multicellular rosettes organize neuropil formation

Brittin, C. A. — 2020-05-28

Neuropils are compartments in the nervous system containing dense networks of neurites and synapses which function as information processing centers. Neuropil formation requires structural and functional organization at and across different scales, achieving single-axon precision for circuits that carry out the core functions while simultaneously accommodating variability among individuals [1; 2; 3; 4]. How these organizational features emerge over development is poorly understood. The nerve ring is the primary neuropil in C. elegans, and its structure is thoroughly mapped [5; 6]. We show that prior to axon outgrowth, nerve ring neurons form a ring of multicellular rosettes with surrounding cells to organize the stratified nerve ring structure [7; 8]. Axon bundles which correspond to future nerve ring strata grow from rosette centers, travel along the ring on "bridge" cells that are simultaneously engaged in adjacent rosettes, and assemble into a topographic scaffold of the nerve ring. SAX-3/Robo is required for proper rosette formation and outgrowth from the center. Furthermore, axon contact sites that form early in development are more conserved than the later ones, indicating a temporal component in neuropil structural variability. Our results reveal an unexpected and critical role of collective cell behaviors prior to innervation to pattern a complex neuropil and orchestrate its formation across scales.

ALX1-related Frontonasal Dysplasia Results From Defective Neural Crest Cell Development and Migration

Pini, J. — 2020-06-13

A pedigree of subjects with frontonasal dysplasia (FND) presented with bilateral oblique facial clefts and ocular phenotypes. Genome sequencing and analysis identified a L165F missense variant in the homeodomain of the transcription factor ALX1 which was imputed to be pathogenic. Induced pluripotent stem cells (iPSC) were derived from the subjects and differentiated to neural crest cells (NCC). NCC derived from ALX1L165F/L165F iPSC were more sensitive to apoptosis, showed an elevated expression of several neural crest progenitor state markers, and exhibited impaired migration compared to wild type controls. NCC migration was also evaluated in vivo using lineage tracing in a zebrafish model, which revealed defective migration of the anterior NCC stream that contributes to the median portion of the anterior neurocranium, phenocopying the clinical presentation. Analysis of human NCC culture media revealed a change in the level of bone morphogenic proteins (BMP), with a low-level of BMP2 and a high level of BMP9. Soluble BMP2 and BMP9 antagonist treatments were able to rescue the defective migration phenotype. Taken together, these results demonstrate a mechanistic requirement of ALX1 in NCC development and migration.

Specific ZNF274 binding interference at SNORD116 activates the maternal transcripts in Prader-Willi syndrome neurons

Langouet, M. — 2020-06-14

Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia, developmental delay, and hyperphagia/obesity. This disorder is caused by the absence of paternally-expressed gene products from chromosome 15q11-q13. We previously demonstrated that knocking out ZNF274, a KRAB-domain zinc finger protein capable of recruiting epigenetic machinery to deposit the H3K9me3 repressive histone modification, can activate expression from the normally silent maternal allele of SNORD116 in neurons derived from PWS iPSCs. However, ZNF274 has many other targets in the genome in addition to SNORD116. Depleting ZNF274 will surely affect the expression of other important genes and disrupt other pathways. Here we used CRISPR/Cas9 to delete ZNF274 binding sites at the SNORD116 locus to determine whether activation of the maternal copy of SNORD116 could be achieved without altering ZNF274 protein levels. We obtained similar activation of gene expression from the normally silenced maternal allele in neurons derived from PWS iPSCs, compared to ZNF274 knockout, demonstrating that ZNF274 is directly involved in the repression of SNORD116. These results suggest that interfering with ZNF274 binding at the maternal SNORD116 locus is a potential therapeutic strategy for PWS.

Requirement of Irf6 and Esrp1/2 in frontonasal and palatal epithelium to regulate craniofacial and palate morphogenesis in mouse and zebrafish

Carroll, S. H. — 2020-06-14

ABSTRACTOrofacial clefts are among the most common human congenital malformations. Irf6 and Esrp1 are two key genes important for palate development, conserved across vertebrates. In the zebrafish, we found that irf6 regulates the expression of esrp1. Using RNAscope, we detailed overlapping Irf6 and Esrp1/2 gene expression in the mouse frontonasal prominence ectoderm, lambda joint periderm, palate and lip epithelium. In the zebrafish, irf6 and esrp1/2 share expression in the pre-gastrulation periderm and the embryonic frontonasal ectoderm, oral epithelium ventral to the anterior neurocranium (ANC), and the developing stomodeum. Genetic disruption of irf6 and esrp1/2 in the zebrafish resulted in cleft of the ANC. In the esrp1/2 zebrafish mutant, cleft of the mouth opening formed and appeared to tether into the ANC cleft. Lineage tracing of the anterior cranial neural crest cells revealed that cleft of the ANC resulted not from migration defect, but from impaired chondrogenesis. Molecular analysis of the aberrant cells localized within the ANC cleft revealed that this cell population espresses sox10, col1a1 and irf6 and is adjacent to cells expressing epithelial krt4. Detailed morphogenetic analysis of mouse Irf6 mutant revealed mesenchymal defects not observed in the Esrp1/2 mutant. Analysis of breeding compound Irf6;Esrp1;Esrp2 mutant suggests that these genes interact where the triple mutant is not observed. Taken together, these studies highlight the complementary analysis of Irf6 and Esrp1/2 in mouse and zebrafish models and captured an unique aberrant embryonic cell population that contributes to cleft pathogenesis. Future work characterizing this unqiue sox10+, col1a1+, irf6+ cell population will yield additional insight into cleft pathogenesis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

YANG, D. — 2020-06-17

Macrophage scavenger receptor 1 (MSR1) plays an important role in host defense to bacterial infections, M2 macrophage polarization and lipid homeostasis. However, its physiological function in viral pathogenesis remains poorly defined. Herein, we report that MSR1 facilitates vesicular stomatitis virus (VSV) infection in the spinal cord. Msr1-deficient (Msr1-/-) mice presented reduced morbidity and mortality following lethal VSV infection, along with normal viremia and antiviral innate immune responses, compared to Msr1+/- littermates and wild-type mice. Msr1 expression was selectively upregulated in the spinal cord, which was the predominant target of VSV infection. The viral load in the spinal cord was positively correlated with Msr1 expression level and was reduced in Msr1-/- mice. Through its extracellular domain, MSR1 interacted with VSV surface glycoprotein and facilitated its cellular entry. In conclusion, our results demonstrate that MSR1 serves as a cellular entry receptor for VSV and facilitates its infection specifically in the spinal cord.

3D reconstruction of odontoblast processes of the mouse molar and incisor

Shuhaibar, N. — 2020-06-21

Odontoblast processes are thin cytoplasmic projections that extend from the cell body at the periphery of the pulp toward the dentin-enamel junction. The odontoblast processes function in the secretion and assembly of dentin during development, participate in mechanosensation, and aid in dentin repair in mature teeth. Because they are small and densely arranged, their three-dimensional organization is not well documented. To gain further insight into how odontoblast processes contribute to odontogenesis, we used serial section electron microscopy to examine these processes in the predentin region of mouse molars and incisors. In molars, the odontoblast processes are tubular with a diameter of ~1.8 m. The odontoblast processes near the incisor tip are similarly shaped, but those midway between the tip and apex are shaped like plates. The plates are radially aligned and longitudinally oriented with respect to the growth axis of the incisor. The thickness of the plates is approximately the same as the diameter of molar odontoblast processes. The plates have an irregular edge; the average ratio of width (midway in the predentin) to thickness is 2.3 on the labial side and 3.6 on the lingual side. The plate geometry seems likely to be related to the continuous growth of the incisor and may provide a clue as to the mechanisms by which the odontoblast processes are involved in tooth development.

Heterogeneity of murine periosteum osteochondroprogenitors involved in fracture healing

Matthews, B. G. — 2020-06-24

The periosteum is the major source of cells involved in fracture healing. We sought to characterize progenitor cells and their contribution to bone fracture healing. The periosteum is highly enriched for progenitor cells, including Sca1+ cells, CFU-F and label-retaining cells compared to the endosteum and bone marrow. Using lineage tracing, we demonstrate that SMA identifies long-term, slow-cycling, self-renewing osteochondroprogenitors in the adult periosteum that are functionally important for bone formation during fracture healing. In addition, Col2.3CreER-labeled osteoblast cells contribute around 10% of osteoblasts, but no chondrocytes in fracture calluses. Most periosteal osteochondroprogenitors following fracture, can be targeted by SMACreER. Previously identified skeletal stem cell populations were common in periosteum, but contained high proportions of mature osteoblasts. We have demonstrated that the periosteum is highly enriched for skeletal progenitor cells and there is heterogeneity in the populations of cells that contribute to mature lineages during periosteal fracture healing.

Gap junction internalization and processing in vivo: a 3D immuno-electron microscopy study

Norris, R. — 2020-06-30

Gap junctions have well-established roles in cell-cell communication by way of forming permeable intercellular channels. Less is understood about their internalization, which forms double membrane vesicles containing cytosol and membranes from another cell, called connexosomes or annular gap junctions. Here, we systematically studied the fate of connexosomes in intact ovarian follicles. High pressure frozen, serial sectioned tissue was immunogold labeled for Connexin 43. Within a volume of electron micrographs, every labeled structure was categorized and counted. Surface area measurements indicate that large connexosomes undergo fission. Subsequent modifications are separation of inner and outer membranes, loss of Cx43 from the outer membrane, and outward budding of the modified membranes. We also documented several clear examples of organelle transfer from one cell to another by gap junction internalization. We discuss how connexosome formation and processing may be a novel means for gap junctions to mediate cell-cell communication.

Chromosome Compartments on the Inactive X Guide TAD Formation Independently of Transcription during X-Reactivation

Bauer, M. — 2020-07-03

A hallmark of chromosome organization is the partition into transcriptionally active A and repressed B compartments and into topologically associating domains (TADs). Both structures were regarded absent from the inactive X chromosome, but to be re-established with transcriptional reactivation and chromatin opening during X-reactivation. Here, we combine a tailor-made mouse iPSC-reprogramming system and high-resolution Hi-C to produce the first time-course combining gene reactivation, chromatin opening and chromosome topology during X-reactivation. Contrary to previous observations, we uncover A/B-like compartments on the inactive X harboring multiple subcompartments. While partial X-reactivation initiates within a compartment rich in X-inactivation escapees, it then occurs rapidly along the chromosome, coinciding with acquisition of naive pluripotency, leading to downregulation of Xist. Importantly, we find that TAD formation precedes transcription, suggesting them to be causally independent. Instead, TADs form first within Xist-poor compartments, establishing Xist as common denominator, opposing both gene reactivation and TAD formation through separate mechanisms. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/177790v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1e5ba28org.highwire.dtl.DTLVardef@120ad95org.highwire.dtl.DTLVardef@64264borg.highwire.dtl.DTLVardef@10e168a_HPS_FORMAT_FIGEXP M_FIG Graphical Summary C_FIG

The Roles of Space and Stochasticity in Computational Simulations of Cellular Biochemistry: Quantitative Analysis and Qualitative Insights

Johnson, M. E. — 2020-07-04

Most of the fascinating phenomena studied in cell biology emerge from interactions among highly organized multi-molecular structures and rapidly propagating molecular signals embedded into complex and frequently dynamic cellular morphologies. For the exploration of such systems, computational simulation has proved to be an invaluable tool, and many researchers in this field have developed sophisticated computational models for application to specific cell biological questions. However it is often difficult to reconcile conflicting computational results that use different simulation approaches (for example partial differential equations versus particle-based stochastic methods) to describe the same phenomenon. Moreover, the details of the computational implementation of any particular algorithm may give rise to quantitatively or even qualitatively different results for the same set of starting assumptions and parameters. In an effort to address this issue systematically, we have defined a series of computational test cases ranging from very simple (bimolecular binding in solution) to moderately complex (spatial and temporal oscillations generated by proteins binding to membranes) that represent building blocks for comprehensive three-dimensional models of cellular function. Having used two or more distinct computational approaches to solve each of these test cases with consistent parameter sets, we generally find modest but measurable differences in the solutions of the same problem, and a few cases where significant deviations arise. We discuss the strengths and limitations of commonly used computational approaches for exploring cell biological questions and provide a framework for decision-making by researchers wishing to develop new models for cell biology. As computational power and speed continue to increase at a remarkable rate, the dream of a fully comprehensive computational model of a living cell may be drawing closer to reality, but our analysis demonstrates that it will be crucial to evaluate the accuracy of such models critically and systematically.

Single-cell multimodal glioma analyses reveal epigenetic regulators of cellular plasticity and environmental stress response

Johnson, K. C. — 2020-07-23

Glioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to universal therapeutic resistance. Here, we integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes, and bulk multi-omic profiles across 11 adult IDH-mutant or IDH-wild-type gliomas to delineate sources of intratumoral heterogeneity. We found that local DNA methylation instability, or epimutation burden, was elevated in more aggressive tumors, reflected intratumoral variability, linked with transcriptional disruption, and associated with environmental stress response. We show that the activation of cell-state specific transcription factors is impacted by epimutations and that loosened epigenetic control may facilitate cellular plasticity. Our analyses support that somatic copy number alterations (SCNAs) promote epigenetic instability and that SCNAs largely precede epigenetic and transcriptomic diversification during glioma evolution. We confirmed the link between genetic and epigenetic instability by analyzing larger cohorts of bulk longitudinally collected and spatially separated DNA methylation data. Increased DNA methylation instability was associated with accelerated disease progression, and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work provides an integrative framework to better understand glioma evolution and highlights the importance of epigenetic heterogeneity in shaping therapeutic response.

A Multi-Phenotype System to Discover Therapies for Age-Related Dysregulation of the Immune Response to Viral Infections

White, B. W. — 2020-07-30

Age-related immune dysregulation contributes to increased susceptibility to infection and disease in older adults. We combined high-throughput laboratory automation with machine learning to build a multi-phenotype aging profile that models the dysfunctional immune response to viral infection in older adults. From a single well, our multi-phenotype aging profile can capture changes in cell composition, physical cell-to-cell interaction, organelle structure, cytokines, and other hidden complexities contributing to age-related dysfunction. This system allows for rapid identification of new potential compounds to rejuvenate older adults immune response. We used our technology to screen thousands of compounds for their ability to make old immune cells respond to viral infection like young immune cells. We observed beneficial effects of multiple compounds, of which two of the most promising were disulfiram and triptonide. Our findings indicate that disulfiram could be considered as a treatment for severe coronavirus disease 2019 and other inflammatory infections.

Bone Marrow Stromal Cells in a Mouse Model of Implant Osseointegration

Vesprey, A. — 2020-08-17

Metal implants are commonly used in orthopaedic surgery. The mechanical stability and longevity of implants depend on adequate bone deposition along the implant surface. The cellular and molecular mechanisms underlying peri-implant bone formation (i.e. osseointegration) are incompletely understood. Herein, our goal was to determine the specific bone marrow stromal cell populations that contribute to bone formation around metal implants. To do this, we utilized a mouse tibial implant model that is clinically representative of human joint replacement procedures. Using a lineage-tracing approach with the Acta2.creERT2 and Tmem100.creERT2 transgenic alleles, we found that Pdgfra- and Ly6a/Sca1-expressing stromal cells (PS cells) multiply and differentiate in the peri-implant environment to give rise to osteocytes in newly formed bone tissue. Single cell RNA-seq analysis indicated that PS cells are quiescent in uninjured bone tissue; however, they express markers of proliferation and osteogenic differentiation shortly after implantation surgery. Our findings indicate that PS cells are mobilized to repair bone tissue and facilitate implant osseointegration following surgery. Biologic therapies targeting PS cells might improve osseointegration in patients undergoing orthopaedic procedures.

Cis-Regulatory Differences Explaining Evolved Levels of Endometrial Invasibility in Eutherian Mammals

Suhail, Y. — 2020-09-05

Eutherian (placental) mammals exhibit great differences in the degree of placental invasion into the maternal endometrium, with humans being on the most invasive end. Previously, we have shown that these differences in invasiveness is largely controlled by the stromal fibroblasts of the maternal endometrium, with secondary effect on stroma of other tissues resulting in correlated differences in cancer malignancy. Here, we present a statistical investigation of the second dogma linking the phenotypic and transcriptional differences to the genomic changes across species, revealing the regulatory genomic sequence differences underlying these inter-species differences. We show that gain or loss of specific transcription factor binding site sequences are connected to the inter-species gene-expression differences in a statistically significant manner, with a particularly larger effect on stromal genes related to invasibility. We also uncover transcriptional factors differentially regulating genes related to pro- and anti- invasible property of stroma. This work extends the understanding of inter-species differences in stromal invasion to the causal genomic sequence differences paving new avenues to target stromal characteristics to regulate placental, or cancer invasion.

The phosphatase inhibitor LB-100 acts synergistically with the NPR2 agonist BMN-111 to improve bone growth

Shuhaibar, L. C. — 2020-09-11

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP (cGMP) in chondrocytes and severe short stature, causing achondroplasia (ACH) and acrosomelic dysplasia type Maroteaux, respectively. Previously we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+), and that in control growth plate chondrocytes, FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein. Here we tested whether a phosphatase inhibitor (LB-100) could enhance bone growth in ACH. In ex vivo imaging experiments using a FRET sensor to measure cGMP production in chondrocytes of living tibias from newborn mice, LB-100 counteracts the FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, LB-100 in combination with BMN-111 increases the rate of femur growth by [~]25% vs BMN-111 alone, restores chondrocyte terminal differentiation, increases the proliferative growth plate area of the femur, and reduces the activity of the MAP kinase pathway. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/288589v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1d0f5d3org.highwire.dtl.DTLVardef@1bb0724org.highwire.dtl.DTLVardef@1d206d1org.highwire.dtl.DTLVardef@1bd9ad7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Proteoglycan 4 (PRG4) expression and functionin dry eye associated inflammation

Menon, N. G. — 2020-10-01

PurposeDry eye disease (DED) affects hundreds of millions worldwide. Proteoglycan 4 (PRG4) has been shown to improve signs and symptoms of DED in humans. The objectives of this study were to characterize endogenous PRG4 expression by telomerase-immortalized human corneal epithelial (hTCEpi) cells, examine how exogenous recombinant human PRG4 (rhPRG4) modulates cytokine and chemokine secretion in response to TNF and IL-1{beta}, explore rhPRG4 as a potential substrate and/or inhibitor of MMP-9, and to understand how experimental dry (EDE) in mice affects PRG4 expression. MethodsPRG4 secretion was quantified by Western blotting and PRG4 expression by immunocytochemistry. Cytokine/chemokine release was measured by ELISA, and MMP-9 inhibition was quantified using an MMP-9 inhibitor kit. EDE was induced in mice, and PRG4 was visualized by immunohistochemistry in the cornea and Western blotting in lacrimal gland lysate. ResultshTCEpi cells synthesize and secrete PRG4 in vitro, which is inhibited by TNF and IL-1{beta}. TNF and IL-1{beta} significantly increased secretion of cytokine IL-6 and chemokines IL-8, IP-10, RANTES, and ENA-78, and several of these chemokines were downregulated after cotreatment with rhPRG4. Fluorescently-labelled rhPRG4 was internalized by hTCEpi cells. rhPRG4 was not digested by MMP-9 and inhibited in vitro activity of exogenous MMP-9 both in solution and in the presence of human tears. Finally, EDE decreased corneal and lacrimal gland expression of PRG4. ConclusionsThese results demonstrate rhPRG4s anti-inflammatory properties in the corneal epithelium and its contribution to ocular surface homeostasis, furthering our understanding of PRG4s immunomodulatory properties in the context of DED inflammation.

Cytonemes with complex geometries and composition extend into invaginations of target cells

Kornberg, T. B. — 2020-10-09

Cytonemes are specialized filopodia that mediate paracrine signaling in Drosophila and other animals. Studies using fluorescence confocal microscopy (CM) established their general paths, cell targets, and essential roles in signaling. To investigate details unresolvable by CM, we used high pressure freezing and electron microscopy to visualize cytoneme structures, paths, contents, and contacts. We observed cytonemes previously seen by CM in the Drosophila wing imaginal disc system, including in disc, tracheal air sac primordium (ASP), and myoblast cytonemes, and identified cytonemes extending into invaginations of target cells and cytonemes connecting ASP cells and connecting myoblasts. Diameters of cytoneme shafts vary between wide (206 {+/-} 51.8 nm) and thin (55.9 {+/-}16.2 nm) segments at regular intervals. Actin, ribosomes, and membranous compartments are present throughout; rough endoplasmic reticulum and mitochondria are in wider proximal sections. These results reveal novel structural features of filopodia and provide a basis for understanding cytoneme cell biology and function. SummaryCytoneme signaling filopodia of Drosophila cells have regions defined by oscillating diameters, and contents that include ER, mitochondria and ribosomes, and form contacts along invaginations of target cell membranes.

The FUR-like regulators PerRA and PerRB control a complex signaling network required for mammalian host-adaptation and virulence of Leptospira interrogans

Grassmann, A. A. — 2020-11-05

Leptospira interrogans, the causative agent of most cases of human leptospirosis, must respond to myriad environmental signals during its free-living and pathogenic lifestyles. Previously, we compared L. interrogans cultivated in vitro and in vivo using a dialysis membrane chamber (DMC) peritoneal implant model. From these studies emerged the importance of genes encoding the Peroxide responsive regulators PerRA and PerRB. First described in in Bacillus subtilis, PerRs are widespread in Gram-negative and -positive bacteria, where regulate the expression of gene products involved in detoxification of reactive oxygen species and virulence. Using perRA and perRB single and double mutants, we establish that L. interrogans requires at least one functional PerR for infectivity and renal colonization in a reservoir host. Our finding that the perRA/B double mutant survives at wild-type levels in DMCs is noteworthy as it demonstrates that the loss of virulence is not due to a metabolic lesion (i.e., metal starvation) but instead reflects dysregulation of virulence-related gene products. Comparative RNA-Seq analyses of perRA, perRB and perRA/B mutants cultivated within DMCs identified 106 genes that are dysregulated in the double mutant, including ligA, ligB and lvrA/B sensory histidine kinases. Decreased expression of LigA and LigB in the perRA/B mutant was not due to loss of LvrAB signaling. The majority of genes in the perRA and perRB single and double mutant DMC regulons were differentially expressed only in vivo, highlighting the importance of host signals for regulating gene expression in L. interrogans. Importantly, the PerRA, PerRB and PerRA/B DMC regulons each contain multiple genes related to environmental sensing and/or transcriptional regulation. Collectively, our data suggest that PerRA and PerRB are part of a complex regulatory network that promotes host adaptation by L. interrogans within mammals. Author SummaryLeptospirosis is a neglected tropical disease with a worldwide distribution. Globally, ~1 million cases and ~60,000 deaths are reported each year. The majority of cases of human leptospirosis are associated with Leptospira interrogans. Infection begins when a naive reservoir (or incidental) host comes into direct or indirect contact with urine from an infected reservoir host. While infection in reservoir hosts, including rats and mice, is generally asymptomatic, incidental hosts, including humans, may develop clinical symptoms ranging from mild flu-like illness to fulminant disease. The gene products required by leptospires for infection remain poorly understood. Herein, we establish that the FUR family regulators PerRA and PerRB function in parallel, contributing to infectivity and renal colonization in mice. By comparative transcriptomics, we identified >100 genes that were dysregulated in the perRA/B double mutant cultivated in rat peritoneal cavities, including the virulence determinants LigA and LigB. Importantly, the PerRA, PerRB and PerRA/B DMC regulons contain multiple genes related to environmental sensing and/or transcriptional regulation. Our data suggest that PerRA and PerRB are part of a complex regulatory network that promotes host adaptation by L. interrogans within mammals.

SBGN Bricks Ontology as a tool to describe recurring concepts in molecular networks

Rougny, A. — 2020-11-17

A comprehensible representation of a molecular network is key to communicating and understanding scientific results in systems biology. The Systems Biology Graphical Notation (SBGN) has emerged as the main standard to represent such networks graphically. It has been implemented by different software tools, and is now largely used to communicate maps in scientific publications. However, learning the standard, and using it to build large maps, can be tedious. Moreover, SBGN maps are not grounded on a formal semantic layer and therefore do not enable formal analysis. Here, we introduce a new set of patterns representing recurring concepts encountered in molecular networks, called SBGN bricks. The bricks are structured in a new ontology, the BricKs Ontology (BKO), to define clear semantics for each of the biological concepts they represent. We show the usefulness of the bricks and BKO for both the template-based construction and the semantic annotation of molecular networks. The SBGN bricks and BKO can be freely explored and downloaded at sbgnbricks.org.

The interaction of p38 with its upstream kinase MKK6

Senthil Kumar, G. — 2020-11-27

Mitogen-activated protein kinase (MAPK; p38, ERK and JNK) cascades are evolutionarily conserved signaling pathways that regulate the cellular response to a variety of extracellular stimuli, such as growth factors and interleukins. The MAPK p38 is activated by its specific upstream MAPK kinases, MKK6 and MKK3. However, a comprehensive molecular understanding of how these cognate upstream kinases bind and activate p38 is still missing. Here, we combine NMR spectroscopy and isothermal titration calorimetry to define the binding interface between full-length MKK6 and p38. We show that p38 engages MKK6 not only via its hydrophobic docking groove, but also helix F, a secondary structural element that plays a key role in organizing the kinase core. We also show that, unlike MAPK phosphatases, the p38 conserved docking (CD) site is much less affected by MKK6 binding. Finally, we demonstrate that these interactions with p38 are conserved independent of the MKK6 activation state. Together, our results reveal differences between specificity markers of p38 regulation by upstream kinases, which do not effectively engage the CD site, and downstream phosphatases, which require the CD site for productive binding.

1H, 15N and 13C sequence specific backbone assignment of the MAP Kinase Binding Domain of the Dual Specificity Phosphatase 1 and its interaction with the MAPK p38

Senthil Kumar, G. — 2020-11-27

The sequence-specific backbone assignment of the mitogen-activated protein kinase (MAPK) binding domain of the dual-specificity phosphatase 1 (DUSP1) has been accomplished using a uniformly [13C,15N]-labeled protein. These assignments will facilitate further studies of DUSP1 in the presence of inhibitors/ligands to target MAPK associated diseases and provide further insights into the function of dual-specificity phosphatase 1 in MAPK regulation.

Convolutional neural network for automated mass segmentation in mammography

Abdelhafiz, D. — 2020-12-02

BackgroundAutomatic segmentation and localization of lesions in mammogram (MG) images are challenging even with employing advanced methods such as deep learning (DL) methods. We developed a new model based on the architecture of the semantic segmentation U-Net model to precisely segment mass lesions in MG images. The proposed end-to-end convolutional neural network (CNN) based model extracts contextual information by combining low-level and high-level features. We trained the proposed model using huge publicly available databases, (CBIS-DDSM, BCDR-01, and INbreast), and a private database from the University of Connecticut Health Center (UCHC). ResultsWe compared the performance of the proposed model with those of the state-of-the-art DL models including the fully convolutional network (FCN), SegNet, Dilated-Net, original U-Net, and Faster R-CNN models and the conventional region growing (RG) method. The proposed Vanilla U-Net model outperforms the Faster R-CNN model significantly in terms of the runtime and the Intersection over Union metric (IOU). Training with digitized film-based and fully digitized MG images, the proposed Vanilla U-Net model achieves a mean test accuracy of 92.6%. The proposed model achieves a mean Dice coefficient index (DI) of 0.951 and a mean IOU of 0.909 that show how close the output segments are to the corresponding lesions in the ground truth maps. Data augmentation has been very effective in our experiments resulting in an increase in the mean DI and the mean IOU from 0.922 to 0.951 and 0.856 to 0.909, respectively. ConclusionsThe proposed Vanilla U-Net based model can be used for precise segmentation of masses in MG images. This is because the segmentation process incorporates more multi-scale spatial context, and captures more local and global context to predict a precise pixel-wise segmentation map of an input full MG image. These detected maps can help radiologists in differentiating benign and malignant lesions depend on the lesion shapes. We show that using transfer learning, introducing augmentation, and modifying the architecture of the original model results in better performance in terms of the mean accuracy, the mean DI, and the mean IOU in detecting mass lesion compared to the other DL and the conventional models.

Primary cilia sensitize insulin receptor-mediated negative feedback in pancreatic β cells

Li, Y. — 2020-12-02

Insulin receptors (IR) can localize to the primary cilia of pancreatic {beta} cells. Because primary cilia are known to sensitize or bias the signaling of cell surface receptors, we investigate how ciliary insulin receptors influence glucose-stimulated insulin secretion (GSIS) in {beta} cells by gauging how cytosolic calcium concentration changes in a mouse insulinoma cell line (MIN6). Purified recombinant insulin suppresses calcium elevation in response to glucose in these cells. Interestingly, ciliated cells show attenuated cytosolic calcium elevation compared to cilium-free cells after glucose stimulation even in the absence of exogenous insulin. We observe that ciliary IR is highly phosphorylated, and the phospho-IR density decreases when cells are either treated with an insulin receptor (IR) inhibitor, BMS536924, or ciliary function is disrupted through either IFT88 or BBS1 knockdown. Consistently, the attenuation of calcium elevation in ciliated cells is abrogated when cells are either treated with IR inhibitor or when primary cilia are impaired. We further demonstrate that ciliary IR signaling hyperpolarizes the plasma membrane but has no apparent impact on glucose-induced ATP production. Thus, our results argue that primary cilia sensitize insulin receptor signaling and mediate negative feedback in GSIS in pancreatic {beta} cells.

Solubility product constant directs the formation of biomolecular condensates

Chattaraj, A. — 2020-12-27

Biomolecular condensates, formed by liquid-liquid phase separation (LLPS), are important cellular structures. Using stochastic network-free kinetic models, we establish a physical-chemical basis for the concentration threshold of heterotypic multivalent molecules required for LLPS. We associate phase separation with a bimodal partitioning of the cluster distribution into small oligomers vs. huge polymers. The simulations reveal that LLPS obeys the solubility product constant (Ksp): the product of monomer concentrations, accounting for ideal stoichiometries, does not exceed a threshold no matter how much additional monomer is added to the system - additional monomer is funneled into large clusters. The Ksp applies over a range of valencies and stoichiometries. However, consistent with the importance of disordered domains for LLPS, removing flexible linker domains funnels valency-matched monomers into a "dimer trap", and Ksp no longer defines a threshold for large cluster formation. We propose Ksp as a new tool for elucidating biomolecular condensate biophysics.

A read count-based method to detect multiplets and their cellular origins from snATAC-seq data

Thibodeau, A. — 2021-01-05

Similar to other droplet-based single cell assays, single nucleus ATAC-seq (snATAC-seq) data harbor multiplets that confound downstream analyses. Detecting multiplets in snATAC-seq data is particularly challenging due to its sparsity and trinary nature (0 reads: closed chromatin, 1: open in one allele, 2: open in both alleles), yet offers a unique opportunity to infer multiplets when >2 uniquely aligned reads are observed at multiple loci. Here, we implemented the first read count-based multiplet detection method, ATAC-DoubletDetector, that detects multiplets independently of cell-type. Using PBMC and pancreatic islet datasets, ATAC-DoubletDetector captured simulated heterotypic multiplets (different cell-types) with [~]0.60 recall, showing [~]24% improvement over state of the art. ATAC-DoubletDetector detected homotypic multiplets with [~]0.61 recall, representing the first method to detect multiplets originating from the same cell type. Using our novel clustering-based algorithm, multiplets were annotated to their cellular origins with [~]85% accuracy. Application of ATAC-DoubletDetector will improve downstream analysis of snATAC-seq.

Mathematical modeling of the Candida albicans yeast to hyphal transition reveals novel control strategies

Wooten, D. J. — 2021-01-20

Candida albicans, an opportunistic fungal pathogen, is a significant cause of human infections, particularly in immunocompromised individuals. Phenotypic plasticity between two morphological phenotypes, yeast and hyphae, is a key mechanism by which C. albicans can thrive in many microenvironments and cause disease in the host. Understanding the decision points and key driver genes controlling this important transition, and how these genes respond to different environmental signals is critical to understanding how C. albicans causes infections in the host. Here we build and analyze a Boolean dynamical model of the C. albicans yeast to hyphal transition, integrating multiple environmental factors and regulatory mechanisms. We validate the model by a systematic comparison to prior experiments, which led to agreement in 18 out of 22 cases. The discrepancies motivate alternative hypotheses that are testable by follow-up experiments. Analysis of this model revealed two time-constrained windows of opportunity that must be met for the complete transition from the yeast to hyphal phenotype, as well as control strategies that can robustly prevent this transition. We experimentally validate two of these control predictions in C. albicans strains lacking the transcription factor UME6 and the histone deacetylase HDA1, respectively. This model will serve as a strong base from which to develop a systems biology understanding of C. albicans morphogenesis.

Differential roles of RIG-I-like receptors in SARS-CoV-2 infection

Yang, D. — 2021-02-11

The retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) are the major viral RNA sensors that are essential for activation of antiviral immune responses. However, their roles in severe acute respiratory syndrome (SARS)-causing coronavirus (CoV) infection are largely unknown. Herein we investigate their functions in human epithelial cells, the primary and initial target of SARS-CoV-2, and the first line of host defense. A deficiency in MDA5 (MDA5-/-), RIG-I or mitochondrial antiviral signaling protein (MAVS) greatly enhanced viral replication. Expression of the type I/III interferons (IFN) was upregulated following infection in wild-type cells, while this upregulation was severely abolished in MDA5-/- and MAVS-/-, but not in RIG-I-/- cells. Of note, ACE2 expression was ~2.5 fold higher in RIG-I-/- than WT cells. These data demonstrate a dominant role of MDA5 in activating the type I/III IFN response to SARS-CoV-2, and an IFN-independent anti-SARS-CoV-2 role of RIG-I.

Intrinsic cardiac adrenergic cell contributes to septic cardiomyopathy

Yang, D. — 2021-03-02

Occurring independently of cardiac sympathetic nervous system, the intrinsic cardiac adrenergic (ICA) cells have been identified as an important regulator in both of developing and adult cardiac physiological and pathological processes. However, its role in septic cardiomyopathy remains unknown. Herein, we report that lipopolysaccharide (LPS) dose- and time-dependently increased norepinephrine (NE) release from ICA cells, which aggravates myocardial TNF- production and dysfunction. Inhibition of NE synthesis in ICA cells alleviated LPS-elicited cardiac dysfunction as well as TNF- production in Langendorff perfusing hearts. Mechanistically, ICA cell expressed Toll-like receptor 4 (TLR4), activated by LPS, to increase the expression of tyrosine hydroxylase, a key enzyme responsible for NE biosynthesis, via AP-1 binding to its promoter. Surprisingly, LPS-TLR4 signaling triggered no TNF- production in ICA cells due to the elevated Nfkbia and Tnfaip6 expression. In LPS-treated co-culture of ICA cells and cardiomyocytes, the raised NE from ICA cells activated cardiomyocyte {beta}1-adrenergic receptor ({beta}1-AR), driving Ca2+/calmodulin-dependent protein kinase II (CaMKII) to increase the activities of NF-{kappa}B and mitogen-activated protein kinase pathways, which were mimicked by dobutamine. Our findings reveal a cell type-specific TLR4 function triggering NE synthesis, but not TNF- production in inflammatory pathogenesis, and identify ICA cell-derived NE as a paracrine signal in the cross talk among different cardiac cells to enhance myocardial injury during LPS challenge, suggesting that targeting ICA cell-derived NE may be a potential therapeutic strategy for septic cardiomyopathy.

PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107

Fowle, H. — 2021-03-03

Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine Protein Phosphatase 2 (PP2A) holoenzyme, PP2A/B55, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55 recruits p107, a pRB-related tumor suppressor and B55 substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an "HxRVxxV619-625" short linear motif (SLiM) in p107 as necessary for B55 binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, "p[ST]-P-x(5-10)-[RK]-V-x-x-[VI]-R". Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55 groove, and phosphosite presentation. Altogether these data provide key insights into PP2A/B55 mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55[<] role in multiple cellular processes.

UBXN3B Restricts Viral Pathogenesis by Maintaining Hematopoietic Homeostasis

WANG, P. — 2021-03-04

Hematopoiesis is finely regulated to enable timely production of the right numbers and types of mature immune cells to maintain tissue homeostasis. Dysregulated hematopoiesis may compromise antiviral immunity and/or exacerbate immunopathogenesis. Herein, we report an essential role of UBXN3B in maintenance of hematopoietic homeostasis and restriction of immunopathogenesis during respiratory viral infection. Ubxn3b deficient (Ubxn3b-/-) mice are highly vulnerable to SARS-CoV-2 and influenza A infection, characterized by more severe lung immunopathology, lower virus-specific IgG, significantly fewer B cells, but more myeloid cells than Ubxn3b+/+ littermates. This aberrant immune compartmentalization is recapitulated in uninfected Ubxn3b-/- mice. Mechanistically, UBXN3B controls precursor B-I (pre-BI) transition to pre-BII and subsequent proliferation in a cell-intrinsic manner, by maintaining BLNK protein stability and pre-BCR signaling. These results reveal an essential role of UBXN3B for the early stage of B cell development.

runBioSimulations: an extensible web application that simulates a wide range of computational modeling frameworks, algorithms, and formats

Shaikh, B. — 2021-03-05

Comprehensive, predictive computational models have significant potential for science, bioengineering, and medicine. One promising way to achieve more predictive models is to combine submodels of multiple subsystems. To capture the multiple scales of biology, these submodels will likely require multiple modeling frameworks and simulation algorithms. Several community resources are already available for working with many of these frameworks and algorithms. However, the variety and sheer number of these resources make it challenging to find and use appropriate tools for each model, especially for novice modelers and experimentalists. To make these resources easier to use, we developed runBioSimulations (https://run.biosimulations.org), a single web application for executing a broad range of models. runBioSimulations leverages community resources, including BioSimulators, a new open registry of simulation tools. These resources currently enable runBioSimulations to execute nine frameworks and 44 algorithms, and they make runBioSimulations extensible to additional frameworks and algorithms. runBioSimulations also provides features for sharing simulations and interactively visualizing their results. We anticipate that runBioSimulations will foster reproducibility, stimulate collaboration, and ultimately facilitate the creation of more predictive models.

The level of synovial human VEGFA, IL-8 and MIP-1α correlate with truncation of lubricin glycans in osteoarthritis

Huang, S. — 2021-03-11

Osteoarthrithis (OA) is an endemic disease due to the increase of the worlds elderly population. Previously thought to be a consequence of an imbalance between cartilage degradation and biosynthesis, it is now recognized as a disease also involving inflammation, hence influencing the level of inflammatory cytokines, growth factors and chemokines. Lubricin is a mucin type molecule where its OA induced glycosylation truncation propels a deteriorating lubrication of the articular cartilage. The objective of this study was to explore the OA driven truncation of O-linked glycosylation of synovial lubricin and its cross talk with systemic and local (synovial fluid, SF) inflammation. We compared the systemic level of cytokines/chemokine in OA patients and controls plasma with their local level in SF using a 44 plex screen. The level of 27 cytokines and chemokines was consistently measured in both plasma and SF. The data showed that the levels of cytokines and chemokines in OA plasma display limited correlation to their counterpart in SF. The level of synovial IL-8 and MIP-1 and VEGFA in OA patients, but not their plasma level, where the only cytokines that displayed a significant correlation to the observed lubricin O-linked glycosylation truncation. These cytokines were also shown to be upregulated exposing fibroblast like synoviocytes from healthy and OA patients to recombinant lubricin with truncated glycans mainly consisting of Tn-antigens, while lubricin with sialylated and non-sialylated T anigens did not have any effect. The data suggest that truncated glycans of lubricin, as found in OA, promotes the synovial cytokine production and exerebate the local synovial inflammation.

Species and strain cultivation of skin, oral, and gut microbiota

Fleming, E. — 2021-04-01

Genomics-driven discovery of microbial species have provided extraordinary insights into the biodiversity of human microbiota. High resolution genomics to investigate species- and strain-level diversity and mechanistic studies, however, rely on the availability of individual microbes from a complex microbial consortia. Here, we describe and validate a streamlined workflow for cultivating microbes from the skin, oral, and gut microbiota, informed by metagenomic sequencing, mass spectrometry, and strain profiling.

MYC dosage compensation is mediated by miRNA-transcription factor interactions in aneuploid cancer

Acon, M. S. — 2021-04-23

We hypothesize that dosage compensation of critical genes arises from systems-level properties for cancer cells to withstand the negative effects of aneuploidy. We identified several candidate genes in cancer multi-omics data and developed a biocomputational platform to construct a mathematical model of their interaction network with miRNAs and transcription factors, where the property of dosage compensation emerged for MYC and was dependent on the kinetic parameters of its feedback interactions with three micro-RNAs. These circuits were experimentally validated with a novel genetic tug-of-war technique by overexpressing an exogenous MYC leading to over-expression of the three microRNAs involved and down-regulation of endogenous MYC. In addition, MYC overexpression or inhibition of its compensating miRNAs led to dosage-dependent cytotoxicity in MYC-amplified colon cancer cells. Finally, we identified negative correlation of MYC dosage compensation with patient survival in TCGA breast cancer patients, highlighting the potential of this mechanism to prevent aneuploid cancer progression. HighlightsThe systems-level property of gene dosage-compensation emerges in silico in miRNA-transcription factor networks depending on the kinetic parameters of its interactions. We established a criterion to identify compensated candidate genes with low variation in expression despite high copy number variation. BioNetUCR is a novel biocomputational platform to model miRNA-transcription factor interactions We present a novel genetic tug-of-war technique to experimentally validate gene dosage compensation at the transcriptional level. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/440572v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@104a93forg.highwire.dtl.DTLVardef@1039ae9org.highwire.dtl.DTLVardef@1af41c6org.highwire.dtl.DTLVardef@1073e6a_HPS_FORMAT_FIGEXP M_FIG C_FIG

ROMO1 is essential for glucose coupling in the pancreatic beta cell of male mice

Wells, L. — 2021-04-28

Aims/HypothesisReactive oxygen species modulator 1 (ROMO1) is a highly conserved inner mitochondrial membrane protein that senses reactive oxygen species and regulates mitochondrial dynamics. ROMO1 is required for mitochondrial fusion in vitro, and silencing ROMO1 increases sensitivity to cell death stimuli. The physiological role of ROMO1 remains unclear. MethodsTo determine the role of ROMO1 in vivo, we used gene targeting in mice to ablate ROMO1 in the whole mouse and to conditionally knock out ROMO1 in the pancreatic beta cell. Mitochondrial functional analyses were performed on isolated mouse and human islets lacking ROMO1. ResultsWe show that ROMO1 is essential for embryonic development, as ROMO1-null mice die before embryonic day 8.5, earlier than GTPases OPA1 or MFN1/2 that catalyze mitochondrial inner and outer membrane fusion. Knockout of ROMO1 in adult pancreatic beta cells results in impaired glucose homeostasis in young male mice due to an insulin secretion defect. Isolated islets from male, but not female, mice showed impaired glucose-stimulated insulin secretion. While mitochondria from female mice were morphologically normal, mitochondria in Romo1 adult beta cell knockout (RABKO) cells from male mice were swollen and fragmented, with a reduction in mtDNA content. Knockout of ROMO1 did not affect basal respiration in males or females, but deletion of ROMO1 in both sexes in mice and isolated human islets reduced spare respiratory capacity (SRC), which involved the specific loss of respiratory activity at Complex II/SDH. Aging of female ROMO1 KO mice resulted in loss of spare respiratory capacity and glucose intolerance. Conclusions/InterpretationOur data demonstrate that ROMO1 is a key regulator of mitochondrial bioenergetics and SRC and is required for effective nutrient coupling to insulin secretion in the beta cell. These observations point to a critical role for spare respiratory capacity in the maintenance of euglycemia and to the potential for targeting ROMO1-complex II to promote glucose coupling in settings of insulin insufficiency. Research in ContextWhat is already known about this subject? O_LIROMO1 is required for mitochondrial fusion C_LIO_LIGlucose coupling to insulin secretion is accomplished in part via generation of NADH during the oxidation of glycolytic metabolites in the TCA cycle C_LIO_LISpare respiratory capacity is lost in aging C_LI What is the key question? O_LIWhat is the physiological role of ROMO1 in the whole animal and the pancreatic beta cell? C_LI What are the new findings? O_LIROMO1 is essential for mouse development C_LIO_LIROMO1 is required to maintain spare respiratory capacity (SRC) and to promote insulin secretion in the beta cells of mice and humans C_LIO_LIAblation of the Romo1 gene in the pancreatic beta cell leads to glucose coupling defects and glucose intolerance in young males and aged females C_LIO_LIAging highlights the importance of SRC in the beta cell for maintaining euglycemia C_LI How might this impact clinical practice in the foreseeable future? O_LIAging is a significant risk factor for T2D. Human males, but not females, experience a loss of insulin secretion with age; designing strategies that enhance ROMO1 and complex II activity to promote SRC may help to reverse these effects. C_LI

Understanding Lactobacillus paracasei and Streptococcus oralis biofilm interactions through agent-based modeling

Archambault, L. — 2021-04-29

As common commensals residing on mucosal tissues, Lactobacillus species are known to encourage health, while recent findings highlight the pathogenic roles of Streptococcus species in these environments. In this study we used a combination of in vivo imaging experiments and computational modeling to explore biofilm interactions between Streptococcus oralis, an accessory pathogen in oral Candidiasis, and Lactobacillus paracasei, an organism with known probiotic properties. A computational agent-based model was created where the two species only interact by competing for space and nutrients. Quantification of bacterial growth in live biofilms indicated that S. oralis biomass and cell numbers were much lower than predicted by the model. Two subsequent models were then created to examine more complex interactions between these species, one where L. paracasei secretes a surfactant, and another where L. paracasei secretes an inhibitor of S. oralis growth. Further biofilm experiments support the hypothesis that L. paracasei may secrete an inhibitor of S. oralis growth, although they do not exclude that a surfactant could also be involved. This contribution shows how agent-based modeling and experiments can be used in synergy to address multiple species biofilm interactions, with important roles in mucosal health and disease. IMPORTANCEWe previously discovered a role of the oral commensal Streptococcus oralis as an accessory pathogen. S. oralis increases the virulence of Candida albicans infections in murine oral candidiasis and epithelial cell models through mechanisms which promote the formation of tissue-damaging biofilms. Lactobacillus species have known inhibitory effects on biofilm formation of many microbes, including Streptococcus species. Agent-based modeling has great advantages as a means of exploring multifaceted relationships between organisms in complex environments such as biofilms. Here we used an iterative collaborative process between experimentation and modeling to reveal aspects of the mostly unexplored relationship between S. oralis and L. paracasei in biofilm growth. The inhibitory nature of L. paracasei on S. oralis in biofilms may be exploited as a means of preventing or alleviating mucosal fungal infections.

Large-scale CRISPRi and transcriptomics of Staphylococcus epidermidis identify genetic factors implicated in commensal-pathogen lifestyle versatility

Spoto, M. — 2021-04-29

Staphylococcus (S.) epidermidis is a ubiquitous human commensal skin bacterium that is also one of the most prevalent nosocomial pathogens. The genetic factors underlying this remarkable lifestyle plasticity are incompletely understood, much due to the difficulties of genetic manipulation, precluding high-throughput functional profiling of this species. To probe S. epidermdis versatility to survive across a diversity of skin sites and infection niches, we developed a large-scale CRISPR interference (CRISPRi) screen complemented by transcriptional profiling (RNA-seq) across 24 diverse environmental conditions and piloted a droplet-based CRISPRi approach to enhance throughput and sensitivity. We identified putative essential genes, importantly, revealing amino acid metabolism as crucial to survival across diverse environments and demonstrated the importance of trace metal uptake for survival under multiple stress conditions. We identified pathways significantly enriched and repressed across our range of stress and nutrient limited conditions, demonstrating the considerable plasticity of S. epidermidis in responding to environmental stressors. We postulate a mechanism by which nitrogen metabolism is linked to lifestyle versatility in response to hyperosmotic challenges, such as those encountered on human skin. Finally, we examined S. epidermidis survival under acid stress and hypothesize a role for cell wall modification as a vital component of the survival response in acidic conditions. Taken together, this study integrates large scale CRISPRi and transcriptomics data across multiple environments to provide insights into a keystone member of the human skin microbiome. Our results additionally provide a valuable benchmarking analysis for CRISPRi screens and are rich resource for other staphylococcal researchers. Author summaryStaphylococcus epidermidis is an important bacteria of the skin microbiome. While it has an important role in skin health, it can also be a major infectious agent, especially in bloodstream and catheter infections. Understanding the underlying genes and pathways that contribute to S. epidermidis ability to have both health and disease-associated abilities will be important to promoting the former and targeting the latter. Yet the function of many S. epidermidis genes, particularly in skin and infection environments, remains unknown. We developed a CRISPRi platform to knock down the function of S. epidermidis genes to better understand to what degree they are essential for growth in these environments. We complemented this gene essentiality data with gene expression data in the same environments to understand how regulation of these genes contribute to S. epidermidis survival. These large-scale data generated numerous hypotheses for new genetic links to S. epidermidis growth versatility.

Identification of Splice Regulators of Fibronectin-EIIIA and EIIIB by Direct Measurement of Exon Usage in a Flow-Cytometry Based CRISPR Screen

Hensel, J. A. — 2021-05-05

The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.

A Method for Rapid Flow-cytometric Isolation of Endothelial Nuclei and RNA from Archived Frozen Brain Tissue

Kimble, A. L. — 2021-05-13

Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.

Functional characterization of splicing regulatory elements

Adamson, S. I. — 2021-05-14

BackgroundRNA binding protein-RNA interactions mediate a variety of processes including pre-mRNA splicing, translation, decay, polyadenylation and many others. Previous high-throughput studies have characterized general sequence features associated with increased and decreased splicing of certain exons, but these studies are limited by not knowing the mechanisms, and in particular, the mediating RNA binding proteins, underlying these associations. ResultsHere we utilize ENCODE data from diverse data modalities to identify functional splicing regulatory elements and their associated RNA binding proteins. We identify features which make splicing events more sensitive to depletion of RNA binding proteins, as well as which RNA binding proteins act as splicing regulators sensitive to depletion. To analyze the sequence determinants underlying RBP-RNA interactions impacting splicing, we assay tens of thousands of sequence variants in a high-throughput splicing reporter called Vex-seq and confirm a small subset in their endogenous loci using CRISPR base editors. Finally, we leverage other large transcriptomic datasets to confirm the importance of RNA binding proteins which we designed experiments around and identify additional RBPs which may act as additional splicing regulators of the exons studied. ConclusionsThis study identifies sequence and other features underlying splicing regulation mediated specific RNA binding proteins, as well as validates and identifies other potentially important regulators of splicing in other large transcriptomic datasets.

Unsupervised deep learning method for cell segmentation

Ud Din, N. — 2021-05-18

Advances in the artificial neural network have made machine learning techniques increasingly more important in image analysis tasks. Recently, convolutional neural networks (CNN) have been applied to the problem of cell segmentation from microscopy images. However, previous methods used a supervised training paradigm in order to create an accurate segmentation model. This strategy requires a large amount of manually labeled cellular images, in which accurate segmentations at pixel level were produced by human operators. Generating training data is expensive and a major hindrance in the wider adoption of machine learning based methods for cell segmentation. Here we present an alternative strategy that uses unsupervised learning to train CNNs without any human-labeled data. We show that our method is able to produce accurate segmentation models. More importantly, the algorithm is applicable to both fluorescence and bright-field images, requiring no prior knowledge of signal characteristics and requires no tuning of parameters.

High resolution analysis of proteome dynamics during Bacillus subtilis sporulation

Tu, Z. — 2021-05-20

Bacillus subtilis vegetative cells switch to sporulation upon nutrient limitation. To investigate the proteome dynamics during sporulation, high resolution time-lapse proteomics was performed in a cell population that was induced to sporulate synchronously. Here, we are the first to comprehensively investigate the changeover of sporulation regulatory proteins, coat proteins and other proteins involved in sporulation and spore biogenesis. Protein co-expression analysis revealed four co-expressed modules (blue, brown, green and yellow). Modules brown and green are upregulated during sporulation and contain proteins associated with sporulation. Module blue, is negatively correlated with modules brown and green, and contained ribosomal and metabolic proteins. Finally, module yellow shows co-expression with the three other modules. Notably, several proteins not belonging to any of the known transcription regulons were identified as co-expressed with modules brown and green. We speculate that they may also play roles during sporulation. Finally, levels of some coat proteins, for example morphogenetic coat proteins, decreased late in sporulation. We speculate on their possible role in guiding or helping assembly of other coat proteins, after which they can be disposed of, but such a hypothesis remains to be experimentally addressed.

Acetylated microtubules are required for maintenance of the barrier between two adjacent tissues

Antel, M. — 2021-06-11

Microtubule acetylation is found in populations of stable, long-lived microtubules, occurring on the conserved lysine 40 (K40) residue of -tubulin, catalyzed by -tubulin acetyltransferases (TATs). K40 acetylation has been shown to stabilize microtubules via enhancing microtubule resilience against mechanical stress. Here we show that Drosophila CG17003/leaky (Lky), an TAT, is required for proper oogenesis. We found that loss of lky disrupted the cell junction between germline cyst and follicle epithelial cells, adjacent cells that form an egg chamber. This resulted in leakage of germline contents into somatic follicle cells. The follicle cells that received germline-derived nanos gene product failed to maintain their cell fate, leading to an egg chamber fusion. The same phenotype was observed upon replacement of major -tubulin84BK40 with -tubulin84BK40A (non-acetylable tubulin), suggesting -tubulinK40 acetylation is required for the boundary integrity of these two adjacent tissues. Taken together, this study provides the first in vivo function of tubulin acetylation in maintaining the integrity of a tissue barrier.

A CRISPR toolbox for generating intersectional genetic mice for functional, molecular, and anatomical circuit mapping

Lusk, S. — 2021-06-10

BackgroundA full understanding of circuits and cellular mechanisms governing health and disease requires the dissection and multi-faceted study of discrete cell subtypes in developing and adult animal models. Recombinase-driven expression of transgenic response alleles represents a significant and powerful approach to delineate cell populations for functional, molecular, and anatomical study. In addition to single recombinase systems, the expression of two recombinases in distinct, but partially overlapping, populations allow for more defined target expression. Although the application of this method is becoming increasingly popular, the expense and difficulty associated with production of customized intersectional mouse lines have limited widespread application to more common allele manipulations that are often commercially produced at great expense. ResultsWe present a simplified CRISPR toolkit for rapid, inexpensive, and facile intersectional allele production. Briefly, we produced 7 intersectional mouse lines using a dual recombinase system, one mouse line with a single recombinase system, and three embryonic stem (ES) cell lines that are designed to study how functional, molecular, and anatomical features relate to each other in building circuits that underlie physiology and behavior. As a proof-of-principle, we applied three of these lines to different neuronal populations for anatomical mapping and functional in vivo investigation of respiratory control. We also generated a mouse line with a single recombinase-responsive allele that controls the expression of the calcium sensor Twitch-2B. This mouse line was applied globally to study the effects of follicle stimulating hormone (FSH) and luteinizing hormone (LH) on calcium release in the ovarian follicle. ConclusionsLines presented here are representative examples of outcomes possible with the successful application of our genetic toolkit for the facile development of diverse, modifiable animal models. This toolkit will allow labs to create single or dual recombinase effector lines easily for any cell population or subpopulation of interest when paired with the appropriate Cre and FLP recombinase mouse lines or viral vectors. We have made our tools and derivative intersectional mouse and ES cell lines openly available for non-commercial use through publicly curated repositories for plasmid DNA, ES cells, and transgenic mouse lines.

Identification of enamel knot gene signature within the developing mouse molar

Winchester, E. W. — 2021-06-15

In most mammals, the primary teeth develop in utero and the cells capable of contributing to hard surface regeneration are lost before tooth eruption. These cells differentiate through a series of reciprocal induction steps between the epithelium and mesenchyme, initially orchestrated by an epithelial signaling center called the enamel knot. While the factors secreted by this structure are of interest to the dental regeneration and development communities, its small size makes it difficult to isolate for analysis. Here we describe our work to identify the enamel knot from whole E14 molars using publicly available scRNA-seq data. We identified 335 genes differentially expressed in the enamel knot compared to the surrounding tissues, including known enamel knot marker genes. We validated expression of the most highly enriched enamel knot marker genes and identified 42 novel marker genes of the enamel knot which provide excellent targets for future dental regeneration investigations.

Splice Factor Polypyrimidine tract-binding protein 1 (Ptbp1) is Required for Immune Priming of the Endothelium in Atherogenic Disturbed Flow Conditions

Hensel, J. A. — 2021-06-18

NF{kappa}B mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through upregulation of adhesion molecules Icam1 and Vcam. The endothelium is "primed" for cytokine activation of NF{kappa}B by exposure to low and disturbed blood flow (LDF) in vivo and by LDF or static conditions in cultured cells. While priming leads to an exaggerated expression of Icam1 and Vcam following cytokine stimulation, the molecular underpinnings are not fully understood. We showed that alternative splicing of genes regulating NF{kappa}B signaling occurs during priming, but the functional implications of this are not known. We hypothesize that the regulation of splicing by RNA-binding splice factors is critical for priming. Here, we perform a CRISPR screen in cultured aortic endothelial cells to determine whether splice factors active in the response to LDF participate in endothelial cell priming. Using Icam1 and Vcam induction by TNF stimulation as a marker of priming, we identify polypyrimidine tract binding protein (Ptbp1) as a required splice factor. Ptbp1 expression is increased and its motifs are enriched nearby alternatively spliced exons in endothelial cells exposed to LDF in vivo in a platelet dependent manner, indicating its induction by early innate immune cell recruitment. At a mechanistic level, deletion of Ptbp1 inhibited NF{kappa}B nuclear translocation and transcriptional activation. These changes coincided with altered splicing of key components of the NF{kappa}B signaling pathway that were similarly altered in the LDF response. However, these splicing and transcriptional changes could be restored by expression of human PTBP1 cDNA in Ptbp1 deleted cells. In vivo, endothelial specific deletion of Ptbp1 reduced myeloid cell infiltration at regions of LDF in atherosclerotic mice. In human coronary arteries, PTBP1 expression correlates with expression of TNF pathway genes and amount of plaque. Together, our data suggest that Ptbp1, which is activated in the endothelium by innate immune cell recruitment in regions of LDF, is required for priming of the endothelium for subsequent NF{kappa}B activation and myeloid cell recruitment in vascular inflammation. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Plaque forms in low and disturbed flow regions of the vasculature, where endothelial cells are "primed" to respond to cytokines (e.g. TNF) with elevated levels of cell adhesion molecules via the NF{kappa}B signaling pathway. We show that the splice factor Ptbp1 (purple) mediates priming. Ptbp1 is induced in endothelial cells by platelet recruitment, promoting priming and subsequent myeloid cell infiltration into plaque. Mechanistically, Ptbp1 regulates splicing of genes involved in the NF{kappa}B signaling pathway and is required for efficient nuclear translocation of NF{kappa}B in endothelial cells. This provides new insight into the molecular mechanisms underlying an endothelial priming process that reinforces vascular inflammatory responses. C_FIG_DISPLAY

Woman Authorship in Pre-print Versus Peer-Reviewed Oral Health-Related Publications: A Two-Year Observational Study

Ioannidou, E. — 2021-06-26

ObjectivesWomen in oral health science face similar societal issues and challenges as those in other STEMM careers, and gender disparities continue to exist as evidenced by fewer women represented as first and last authors in scientific publications. Pre-prints may serve as a conduit to immediately disseminating ones work, bypassing the arduous peer review process and its associated inherent biases. Therefore, the purpose of this study was to 1] compare the gender of first and last authors in pre-print versus peer reviewed publications, 2] examine the composition of first and last author pairs as stratified by publication type, and 3] examine the correlation between woman authorship and institutional geographic location and publication metrics stratified by publication type. MethodsThe keyword "oral health" was used to search for publications in BioRxiv and Pubmed in the years 2018 and 2019. Gender of first and last authors were determined, and its frequency was considered as the primary outcome. Additionally, the geographic location of the authors associated institution and publication metrics measured by Altmetrics score were extracted. Data was descriptively summarized by frequencies and percentages. Chi-square analysis was conducted for categorical variables which included the relationship between gender and publication type as well as gender and region of authors associated institution. Binomial regression analysis was conducted to analyze the relationship between gender and Altmetrics. ResultsWoman first authors comprised 40.3% of pre-prints and 64.5% of peer reviewed publications [p<0.05]. Woman last authors comprised 31.3% of pre-prints and 61.5% of peer reviewed publications [p<0.05]. When analyzing the relationships between first and last author, the Man-Man pairing represented 47.7% of the pre-print publications and the Woman-Woman pairing comprised a majority of the of the peer review publications at 47.5%. All results were statistically significant with a p-value <0.05. No significant correlation was found between region of institution or Altmetrics and gender of first or last authors [p>0.05]. ConclusionFor the first time in oral health science, it was found that women show higher representation as first and last author positions in peer reviewed publications versus pre-prints.

Early detection of SARS-CoV-2 in circulating immune cells in a mouse model

Geng, T. — 2021-06-30

SARS-CoV-2 infects the respiratory tract, lung and then other organs. However, its pathogenesis remains largely unknown. We used RareScope Fluorescence Light Sheet Microscopy (FLSM) and fluorescent in situ hybridization of RNA (RNA-FISH) to detect SARS-CoV-2 RNA and dissemination kinetics in mouse blood circulation. By RNA-FISH, we found that SARS-CoV-2 RNA-positive leukocytes, including CD11c cells, appeared as early as one day after infection and continued through day 10 post infection. Our data suggest that SARS-CoV-2-permissive leukocytes contribute to systemic viral dissemination, and RNA-FISH combined with FLSM can be utilized as a sensitive tool for SARS-CoV-2 detection in blood specimens.

Antisense oligonucleotides targeting UBE3A-ATS restore expression of UBE3A by relieving transcriptional interference

Germain, N. D. — 2021-07-10

Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by loss of function of the maternally inherited UBE3A allele. In neurons, the paternal allele of UBE3A is silenced in cis by the long noncoding RNA, UBE3A-ATS. Unsilencing paternal UBE3A by reducing UBE3A-ATS is a promising therapeutic approach for the treatment of AS. Here we show that targeted cleavage of UBE3A-ATS using antisense oligonucleotides (ASOs) restores UBE3A and rescues electrophysiological phenotypes in human AS neurons. We demonstrate that cleavage of UBE3A-ATS results in termination of its transcription by displacement of RNA Polymerase II. Reduced transcription of UBE3A-ATS allows transcription of UBE3A to proceed to completion, providing definitive evidence for the transcriptional interference model of paternal UBE3A silencing. These insights into the mechanism by which ASOs restore UBE3A inform the future development of nucleotide-based approaches for the treatment of AS, including alternative strategies for cleaving UBE3A-ATS that can be developed for long-term restoration of UBE3A function.

A model of actin-driven endocytosis explains differences of endocytic motility in budding and fission yeast

Nickaeen, M. M. — 2021-07-21

A comparative study (Sun et al., eLife, 2019) showed that the abundance of proteins at sites of endocytosis in fission and budding yeast is more similar in the two species than previously thought, yet membrane invaginations in fission yeast elongate two-fold faster and are nearly twice as long as in budding yeast. Here we use a three-dimensional model of a motile endocytic invagination (Nickaeen et al., MBoC, 2019) to investigate factors affecting elongation of the invaginations. We found that differences in turgor pressure in the two yeast species can largely explain the paradoxical differences observed experimentally in endocytic motility.

Parental origin of Gsα inactivation differentially affects bone remodeling in a mouse model of Albright hereditary osteodystrophy

McMullan, P. — 2021-07-27

Albright hereditary osteodystrophy (AHO) is caused by heterozygous inactivation of GNAS, a complex locus that encodes the alpha-stimulatory subunit of GPCRs (Gs) in addition to NESP55 and XLs due to alternative first exons. AHO skeletal manifestations include brachydactyly, brachymetacarpia, compromised adult stature, and subcutaneous ossifications. AHO patients with maternally-inherited GNAS mutations develop pseudohypoparathyroidism type 1A (PHP1A) with resistance to multiple hormones that mediate their actions through GPCRs requiring Gs (eg., PTH, TSH, GHRH, calcitonin) and severe obesity. Paternally-inherited GNAS mutations cause pseudopseudohypoparathyroidism (PPHP), in which patients have AHO skeletal features but do not develop hormonal resistance or marked obesity. These differences between PHP1A and PPHP are caused by tissue-specific reduction of paternal Gs expression. Previous reports in mice have shown loss of Gs causes osteopenia due to impaired osteoblast number and function and suggest AHO patients could display evidence of reduced bone mineral density (BMD). However, we previously demonstrated PHP1A patients display normal-increased BMD measurements without any correlation to body mass index or serum PTH. Due to these observed differences between PHP1A and PPHP, we utilized our laboratorys AHO mouse model to address whether Gs heterozygous inactivation by the targeted disruption of exon 1 of Gnas differentially affects bone remodeling based on the parental inheritance of the mutation. Mice with paternally-inherited (GnasE1+/-p) and maternally-inherited (GnasE1+/-m) mutations displayed reductions in osteoblasts along the bone surface compared to wildtype. GnasE1+/-p mice displayed reduced cortical and trabecular bone parameters due to impaired bone formation and excessive bone resorption. GnasE1+/-m mice however displayed enhanced bone parameters due to increased osteoblast activity and normal bone resorption. These distinctions in bone remodeling between GnasE1+/-p and GnasE1+/-m mice appear to be secondary to changes in the bone microenvironment driven by calcitonin-resistance within GnasE1+/-m osteoclasts and therefore warrant further studies into understanding how Gs influences osteoblast-osteoclast coupling interactions.

Single cell analysis of endometriosis reveals a coordinated transcriptional program driving immunotolerance and angiogenesis across eutopic and ectopic tissues.

Tan, Y. — 2021-07-29

Endometriosis is characterized by growth of endometrial-like tissue outside of the uterus affecting many women in their reproductive age, causing years of pelvic pain and potential infertility. Its pathophysiology remains largely unknown, limiting diagnosis and treatment. We characterized peritoneal and ovarian lesions at single-cell transcriptome resolution and compared to matched eutopic endometrium, control endometrium, and organoids derived from these tissues, generating data on over 100,000 cells across 12 individuals. We spatially localized many of the cell types using imaging mass cytometry. We identify a perivascular mural cell unique to the peritoneal lesions with dual roles in angiogenesis promotion and immune cell trafficking. We define an immunotolerant peritoneal niche, fundamental differences in eutopic endometrium and between lesions microenvironments, and a novel progenitor-like epithelial cell subpopulation. Altogether, this study provides a holistic view of the endometriosis microenvironment representing the first comprehensive cell atlas of the disease, essential information for advancing therapeutics and diagnostics.

TRPM2 deficiency protects against atherosclerosis by inhibiting TRPM2-CD36 inflammatory axis in macrophages

Zong, P. — 2021-07-30

Atherosclerosis is the major cause of ischemic heart diseases and ischemic brain stroke, which are the leading causes of mortality worldwide. The central pathological features of atherosclerosis include macrophage infiltration and foam cell formation. However, the detailed mechanisms regulating these two processes remain unclear. Here we show that oxidative stress-activated Ca2+-permeable TRPM2 plays a key role in the pathogenesis of atherosclerosis. Trpm2 deletion produces a potent protective effect against atherosclerosis in ApoE-/- mice fed with a high-fat diet (HFD), as evidenced by reduced atherosclerotic plaque burden, decreased macrophage load and suppressed inflammasome activation in the vessel wall. Moreover, we show that Trpm2 deletion or inhibition reduces oxidized low-density lipoprotein (oxLDL) uptake by macrophages, suppresses macrophage infiltration induced by monocyte chemoattractant protein-1 (MCP1), and prevents the impairment of macrophage emigration caused by oxLDL. Intriguingly, we uncover that activation of CD36, an oxLDL receptor, can promote the activation of TRPM2, and vice versa, the CD36-mediated inflammatory cascade in atherosclerosis is dependent on TRPM2. In transfected HEK293T cells, CD36 ligands oxLDL and TSP1 induce TRPM2 activation in a CD36-dependent manner. Deleting Trpm2 or inhibiting TRPM2 activity in cultured macrophages suppresses the CD36 signaling cascade induced by oxLDL and TSP1. Our studies establish TRPM2-CD36 axis as a new mechanism underlying atherogenesis, and suggest TRPM2 as an effective therapeutic target for atherosclerosis. HIGHLIGHTSO_LITrpm2 deletion protects against atherosclerosis in ApoE-/- mice fed with a high-fat diet (HFD) C_LIO_LITrpm2 deficiency reduces atherosclerotic lesions by minimizing foam cell formation, inhibiting macrophage infiltration and preserving macrophage emigration C_LIO_LITRPM2 activation is required for CD36-induced oxLDL uptake and subsequent inflammatory responses C_LIO_LIThe ligands of CD36, oxLDL and TSP1, activate TRPM2, thereby perpetuating TRPM2-CD36 inflammatory cycle in atherogenesis cascade C_LIO_LIOur data establish TRPM2-CD36 axis as a new atherogenesis mechanism and TRPM2 as a novel therapeutic target for atherosclerosis C_LI O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY TRPM2-mediated Ca2+ signal is essential for CD36 induced oxLDL uptake and atherosclerosis in ApoE-/- mice fed with a high-fat diet (HFD). The activation of CD36 and TRPM2 form a positive feedback loop in atherogenesis. C_FIG_DISPLAY

Functional coupling of TRPM2 and NMDARs exacerbates excitotoxicity in ischemic brain injury

Zong, P. — 2021-07-30

Excitotoxicity caused by NMDA receptors (NMDARs) is a major cause of neuronal death in ischemic stroke. However, past efforts of directly targeting NMDARs have unfortunately failed in clinical ischemic stroke trials. Here we reveal an unexpected mechanism underlying NMDARs-mediated neurotoxicity, which leads to identification of a novel target and development of an effective therapeutic peptide for ischemic stroke. We show that NMDARs excitotoxicity upon ischemic insults is mediated by physical and functional coupling to TRPM2. The physical interaction of TRPM2 with NMDARs results in markedly increase in the surface expression of NMDARs, leading to enhanced NMDAR function and increased neuronal death. We identified a specific NMDAR-interacting domain on TRPM2, and developed a cell-permeable peptide to uncouple TRPM2-NMDARs. The disrupting-peptide protects neurons against ischemic injury in vitro and protects mice against ischemic stroke in vivo. These findings provide an unconventional strategy to eliminate excitotoxic neuronal death without directly targeting NMDARs. HIGHLIGHTSO_LITRPM2 physically and functionally interacts with NMDARs C_LIO_LIInteraction of TRPM2 with NMDARs exacerbates NMDARs extrasynaptic excitotoxicity by increasing NMDARs surface expression during ischemic injury C_LIO_LITRPM2 recruits PKC{gamma} to the interacting complexes to increase NMDARs surface expression C_LIO_LIUncoupling the interaction between TRPM2 and NMDARs with a disrupting peptide (TAT-EE3) protects neurons against ischemic stroke in vitro and in vivo C_LI GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=175 HEIGHT=200 SRC="FIGDIR/small/454247v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@e618c5org.highwire.dtl.DTLVardef@646350org.highwire.dtl.DTLVardef@b4478corg.highwire.dtl.DTLVardef@1c5742f_HPS_FORMAT_FIGEXP M_FIG C_FIG TRPM2 excerbates NMDARs excitotoxicity by physically and functionally interacting with NMDARs. The disrupting pipette TAT-EE3 protects neurons against ischemic injury in vitro and in vivo.

Integrated single-cell transcriptomic and epigenetic analyses of cell-state transition and lineage commitment in the embryonic mouse cerebellum

Farah-Nagham, K. — 2021-08-08

Recent studies using single-cell RNA-seq have revealed cellular heterogeneity in the developing mammalian cerebellum, yet the regulatory logic underlying this cellular diversity remains to be elucidated. Using integrated single-cell RNA and ATAC analyses, we resolved developmental trajectories of cerebellar progenitors and identified putative trans- and cis- elements that control cell state transition. We reverse-engineered gene regulatory networks (GRNs) of each cerebellar cell type. Through in silico simulations and in vivo experiments, we validated the efficacy of GRN analyses and uncovered the molecular control of a newly identified stem zone, the posterior transitory zone (PTZ), which contains multipotent progenitors for granule neurons, Bergmann glia, and choroid plexus epithelium. Importantly, we showed that perturbing cell fate specification of PTZ progenitors causes posterior cerebellar vermis hypoplasia, the most common cerebellar birth defect in humans. Our study provides a foundation for comprehensive studies of developmental programs of the mammalian cerebellum.

A Novel Model for Encephalomyosynangiosis Surgery after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

Paro, M. — 2021-08-17

Background and PurposeNo effective treatment is available for most patients who suffer ischemic stroke. Development of novel treatment options is imperative. The brain attempts to self-heal after ischemic stroke via various mechanism mediated by restored blood circulation in affected region of brain but this process is limited by inadequate angiogenesis or neoangiogenesis. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis with low morbidity in patients with moyamoya disease, reducing risk of stroke. However, EMS, surgery has never been studied as an therapeutic option after ischemic stroke. Here we described a novel procedure and feasibility data for EMS after ischemic stroke in mice. MethodsA 60 mins of middle cerebral artery occlusion (MCAo) was used to induce ischemic stroke in mice. After 3-4 hours of MCAo onset/sham, EMS was performed. Mortality of EMS, MCAo and. MCAo+EMS mice was recorded up to 21 days after surgery. Graft tissue viability was measured using a nicotinamide adenine dinucleotide reduced tetrazolium reductase assay. ResultsEMS surgery after ischemic stroke does not increase mortality compared to stroke alone. Graft muscle tissue remained viable 21 days after surgery. ConclusionsThis novel protocol is effective and well-tolerated, may serve as novel platform for new angiogenesis and thus recovery after ischemic stroke. If successful in mice, EMS can a very feasible and novel treatment option for ischemic stroke in humans.

A Cellular Reference Resource for the Mouse Urinary Bladder

Baker, D. — 2021-09-23

The urinary bladder functions as a reservoir to store and extrude liquid bodily waste. Significant debate exists as to this tissues cellular composition and genes associated with their functions. We use a repertoire of cell profiling tools to comprehensively define and spatial resolve cell types. We characterize spatially validated, basal-to-luminal gene expression dynamics within the urothelium, the cellular source of most bladder cancers. We define three distinct populations of fibroblasts that spatially organize from the sub-urothelial layer through to the detrusor muscle, clarifying knowledge around these controversial interstitial cells, and associate increased fibroblasts with aging. We overcome challenges of profiling the detrusor muscle, absence from earlier single cell studies, to report on its transcriptome with many novel and neuronal-like features presumably associated with neuromuscular junctions. Our approach provides a blueprint for tissue atlas construction and the data provides the foundation for future studies of bladder function in health and disease.

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore sequencing

Begik, O. — 2021-09-22

RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here, we develop Nanopore 3 end-capture sequencing (Nano3P-seq), a novel method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition and tail length dynamics at per-read resolution. By employing a template switching-based sequencing protocol, Nano3P-seq can sequence any given RNA molecule from its 3 end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq captures a wide diversity of RNA biotypes, providing quantitative estimates of RNA abundance and tail lengths in mRNA, lncRNA, sn/snoRNA, scaRNA, and rRNA molecules. We find that, in addition to mRNA and lncRNA, polyA tails can be identified in 16S mitochondrial rRNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we identify non-A bases within polyA tails of various lengths and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

The connection between Rap1 and Talin1 in CD4+ T Lymphocytes

Lagarrigue, F. — 2021-09-23

Agonist induced increase in integrin affinity for ligands (activation) plays a pivotal role in T cell trafficking and functions. Activation requires Rap1 GTPase-mediated recruitment of talin1 to the integrins in the plasma membrane. Rap1-interacting adaptor molecule (RIAM) is a Rap1 effector that serves this function in T cells. In addition, Rap1 directly binds to talin1 to enable integrin activation in platelets. Here, we assessed the relative contributions of the Rap1-talin1 interaction and RIAM and provide a complete accounting of the connections between Rap1 and talin1 that support integrin activation in conventional CD4+ (Tconv) and CD25HiFoxp3+CD4+ regulatory T (Treg) cells. Disruption of both Rap1 binding sites in talin1 (talin1 (R35E,R118E)) causes a partial defect in L{beta}2, 4{beta}1 and 4{beta}7 integrin activation in both Tconv and Treg cells with resulting defects in T cell homing and functions. Over-expression of RIAM bypasses the integrin activation defect in Tconv cells expressing talin1 (R35E,R118E), indicating that RIAM can substitute for Rap1 binding to talin in integrin activation. Conversely, deletion of RIAM in talin1 (R35E,R118E) Tconv cells abrogates activation of L{beta}2, 4{beta}1 and 4{beta}7. RIAM and lamellipodin (LPD) are mammalian members of the MRL protein family; LPD plays a more important role than RIAM in Treg cell integrin activation. Nevertheless, loss of RIAM profoundly exacerbates the defects in Treg cell function caused by the talin1 (R35E,R118E) mutation. Most importantly, deleting both MRL proteins combined with talin1 (R35E,R118E) phenocopies the complete lack of integrin activation observed in Rap1a/b null Treg cells. In sum, these data reveal the functionally significant connections between Rap1 and talin1 that enable L{beta}2, 4{beta}1 and 4{beta}7 integrin activation in T cells.

Patient-Derived Organoids Recapitulate Intrinsic Immune Landscapes and Progenitor Populations of Glioblastoma.

Watanabe, F. — 2021-10-09

Glioblastoma stem cells (GSCs) are highly self-renewing, resistant to therapy, and are able to form lethal tumors1, 2. Tumor organoids have been developed to study tumor evolution1-4, and while GSCs can form organoids for glioblastoma multiforme, our understanding of their intrinsic immune, metabolic, genetic, and molecular programs is limited. To address this, we deeply characterized GSC-derived GBM organoids using a modified protocol (GBMOsm) from several patient-derived GSCs and found they develop into complex 3D tissues with unique self-organization, cancerous metabolic states, and burdensome genetic landscapes. We discovered that GBMOsc recapitulate the presence of two important cell populations thought to drive GBM progression, SATB2+ and HOPX+ progenitors. Despite being devoid of immune cells, transcriptomic analysis across GBMOsc revealed an immune-like molecular program, enriched in cytokine, antigen presentation and processing, T-cell receptor inhibitors, and interferon genes. We determined that SATB2+ and HOPX+ populations contribute to this immune and interferon landscape in GBM in vivo and GBMOsm. Our work deepens our understanding of the intrinsic molecular and cellular architecture of GSC-derived GBMO and defines a novel GBMOsc intrinsic immune-like program.

Retroactive analysis of single cell transcriptome profiles using next-generation algorithms revised the identification of several resilient retinal ganglion cell types

Rheaume, B. A. — 2021-10-16

The clustering of single cell RNA-sequencing (scRNA-seq) data enables the classification of cell types, and the development of integration mapping algorithms has enabled the tracing of altered-from-baseline transcriptomes to their respective cell type origins. Here, we developed an algorithm that removes sources of noise from scRNA-seq reference dataset, and in the next step optimizes weight-assignment to anchors, cumulatively improving the accuracy of query cells mapping to reference dataset. The denoising step of our algorithm also improved the performance of other mapping algorithms. To further demonstrate biological relevance, using our algorithm we determined the type-origin of the 17% of injured retinal ganglion cells (RGCs) that a prior algorithm did not identify. As we found that most of the originally unassigned cells belonged to only some RGC types, a consequent change in the proportions of the surviving types resulted in an amended ranking of resiliency to injury. We also identified new cluster-markers for RGC types, validated two novel markers by immunostaining in retinas, and developed a website for cluster-by-cluster comparison of gene expression between uninjured and injured RGC types. Additional bioinformatic analyses contributed new insights into the global characteristics of RGC types and how axonal injury affects them, showing how dissimilarity between transcriptomes of RGC types increases during maturation and after injury. We further characterized the correspondence between the neonatal and adult RGC types, and showed which cluster markers change expression developmentally or after injury. We also show, for the first time, that global properties of the transcriptome can predict the resilience to injury of at least some cell types. The R-package, CellTools, for the algorithms we developed, will assist scRNA-seq studies across biological fields.

Post-injury born oligodendrocytes integrate into the glial scar and inhibit growth of regenerating axons by premature myelination

Xing, J. — 2021-10-16

Pathologies of the central nervous system (CNS) white matter often result in permanent functional deficits because mature mammalian projection neurons fail to regenerate long-distance axons after injury. A major barrier to axonal regenerative research is that the CNS axons that regenerate in response to experimental treatments stall growth before reaching their post-synaptic targets. Here, we test the hypothesis that premature, de novo, myelination of regenerating axons stalls their growth, even after bypassing the glial scar. To test this hypothesis, first, we used single cell RNA-seq (scRNA-seq) and immunohistological analysis to investigate whether post-injury born oligodendrocytes integrate into the glial scar after optic nerve injury. Then, we used a multiple sclerosis model of demyelination concurrently with the stimulation of axon regeneration by Pten knockdown (KD) in projection neurons after optic nerve injury. We found that post-injury born oligodendrocytes integrate into the glial scar, where they are susceptible to the demyelination treatment, which prevented premature myelination, and thereby enhanced Pten KD-stimulated axon regeneration. We also present a website for comparing the gene expression of scRNA-seq-profiled optic nerve oligodendrocytes under physiological and pathophysiological conditions. SIGNIFICANCE STATEMENTMyelin debris from degenerating axons along with reactive astrocytes in the glial scar inhibit CNS axon regeneration. However, even with the recently developed experimental approaches which activate axons to regenerate passed the glial scar, almost all axons still stall growth before reaching their post-synaptic targets. Here, we show that post-injury born oligodendrocytes integrate into the glial scar, and that other than myelin debris, live oligodendrocytes prematurely myelinating the regenerating axons inhibit growth, even if the axons have already regenerated passed the glial scar.

Accurate estimation of intrinsic biases for improved analysis of chromatin accessibility sequencing data using SELMA

Hu, S. S. — 2021-10-24

Genome-wide profiling of chromatin accessibility by DNase-seq or ATAC-seq has been widely used to identify regulatory DNA elements and transcription factor binding sites. However, enzymatic DNA cleavage exhibits intrinsic sequence biases that confound chromatin accessibility profiling data analysis. Existing computational tools are limited in their ability to account for such intrinsic biases and not designed for analyzing single-cell data. Here, we present Simplex Encoded Linear Model for Accessible Chromatin (SELMA), a computational method for systematic estimation of intrinsic cleavage biases from genomic chromatin accessibility profiling data. We demonstrate that SELMA yields accurate and robust bias estimation from both bulk and single-cell DNase-seq and ATAC-seq data. SELMA can utilize internal mitochondrial DNA data to improve bias estimation. We show that transcription factor binding inference from DNase footprints can be improved by incorporating estimated biases using SELMA. Furthermore, we show strong effects of intrinsic biases in single-cell ATAC-seq data, and develop the first single-cell ATAC-seq intrinsic bias correction model to improve cell clustering. SELMA can enhance the performance of existing bioinformatics tools and improve the analysis of both bulk and single-cell chromatin accessibility sequencing data.

ANKLE1 cleaves mitochondrial DNA and contributes to cancer risk by driving the Warburg effect and apoptosis resistance

Przanowski, P. — 2021-11-07

Alleles within the chr19p13.1 locus are associated with increased risk of both ovarian and breast cancer and increased expression of the ANKLE1 gene. ANKLE1 is molecularly characterized as an endonuclease that efficiently cuts branched DNA and shuttles between the nucleus and cytoplasm. However, the role of ANKLE1 in mammalian development and homeostasis remains unknown. In normal development ANKLE1 expression is limited to the erythroblast lineage and we found that ANKLE1s role is to cleave the mitochondrial genome during erythropoiesis. We show that ectopic expression of ANKLE1 in breast epithelial-derived cells leads to genome instability and mitochondrial DNA (mtDNA) cleavage. mtDNA degradation then leads to mitophagy and causes a shift from oxidative phosphorylation to glycolysis (Warburg effect). Moreover, mtDNA degradation activates STAT1 and expression of epithelial-mesenchymal transition (EMT) genes. Reduction in mitochondrial content contributes to apoptosis resistance, which may allow precancerous cells to avoid apoptotic checkpoints and proliferate. These findings provide evidence that ANKLE1 is the causal cancer susceptibility gene in the chr19p13.1 locus and describe mechanisms by which higher ANKLE1 expression promotes cancer risk.

Mispatterned motile cilia beating causes flow blockage in the epileptic brain

Faubel, R. J. — 2021-10-30

Beating of motile cilia at the brain ventricular surface generates rapid flow in an evolutionary conserved pattern mediating the transport of cerebrospinal fluid, but its functional importance has yet to be demonstrated. Here we show disturbance of this transport may contribute to seizure susceptibility. Mice haploinsufficient for FoxJ1, transcription factor regulating motile cilia exhibited cilia-driven flow blockage and increased seizure susceptibility. Mutations in two epilepsy-associated kinases, Cdkl5 and Yes1, in mice resulted in similar cilia-driven flow blockage and increased seizure susceptibility. We showed this arises from disorganized cilia polarity associated with disruption in the highly organized basal body anchoring meshwork. Together these findings suggest mispatterning of cilia-generated flow may contribute to epilepsy and thus might account for seizures unresponsive to current seizure medications. One sentence summaryEpilepsy is associated with disturbance of cilia motion and mispatterning of fluid transport in the brain ventricles.

Interchromosomal interaction of homologous Stat92E alleles regulates transcriptional switch during stem-cell differentiation

Antel, M. — 2021-11-08

The strength of pairing of homologous chromosomes differs in a locus-specific manner and is correlated to gene expression states. However, the functional impact of homolog pairing on local transcriptional activity is still unclear. Drosophila male germline stem cells (GSCs) constantly divide asymmetrically to produce one GSC and one differentiating gonialblast (GB). The GB then enters the differentiation program in which stem cell specific genes are quickly downregulated. Here we demonstrate that a change in local pairing state of the Stat92E locus is required for the downregulation of the Stat92E gene during differentiation. Using OligoPaint fluorescent in situ hybridization (FISH), we show that the interaction between homologous regions of Stat92E is always tight in GSCs and immediately loosened in GBs. When one of the Stat92E locus was absent or relocated to another chromosome, Stat92E did not pair and failed to downregulate, suggesting that the pairing is required for switching of transcriptional activity. The defect in downregulation of Stat92E was also observed upon knockdown of global pairing or anti-pairing factors. Moreover, the Stat92E enhancer element, but not cis-transcription, is required for the change in pairing state, indicating that it is not a consequence of transcriptional changes. GSCs are known to inherit pre-existing histones H3 and H4, while newly synthesized histones are distributed in GBs. When this histone inheritance was compromised, the change in Stat92E pairing did not occur, suggesting that it is an intrinsically programmed process during asymmetric stem cell division. We propose that the change of local pairing state may be a common process to reprogram gene activity during cell-differentiation.

Ciliary Generation of a Peptidergic Sexual Signal

Luxmi, R. — 2021-11-08

Peptidergic intercellular communication occurs throughout the eukaryotes, and regulates a wide range of physiological and behavioral responses. Cilia are sensory and secretory organelles that both receive information from the environment and transmit signals. Cilia derived vesicles (ectosomes), formed by outward budding of the ciliary membrane, carry enzymes and other bioactive products; this process represents an ancient mode of regulated secretion. Our previous study revealed the presence of the peptide amidating enzyme, peptidylglycine -amidating monooxygenase (PAM), in cilia and its key role in ciliogenesis. Furthermore, PAM and its amidated products are released in ciliary ectosomes from the green alga Chlamydomonas reinhardtii. One amidated product (GATI-amide) serves as a chemotactic modulator for C. reinhardtii gametes, attracting minus gametes while repelling plus gametes. Here we dissect the complex processing pathway that leads to formation of this amidated peptidergic sexual signal specifically on the ectosomes of plus gametes. We also identify a potential prohormone convertase that undergoes domain rearrangement during ectosomal secretion as a substrate for PAM. Analysis of this pathway affords insight into how single-celled organisms lacking dense core vesicles engage in regulated secretion, and provides a paradigm for understanding how amidated peptides that transmit sexual and other signals through cilia are generated.

Kinetic networks identify key regulatory nodes and transcription factor functions in early adipogenesis

Dutta, A. B. — 2021-11-19

Adipocytes contribute to metabolic disorders such as obesity, diabetes, and atherosclerosis. Prior characterizations of the transcriptional network driving adipogenesis overlook transiently acting transcription factors (TFs), genes, and regulatory elements that are essential for proper differentiation. Moreover, traditional gene regulatory networks provide neither mechanistic details about individual RE-gene relationships nor temporal information needed to define a regulatory hierarchy that prioritizes key regulatory factors. To address these shortcomings, we integrate kinetic chromatin accessibility (ATAC-seq) and nascent transcription (PRO-seq) data to generate temporally resolved networks that describe TF binding events and resultant effects on target gene expression. Our data indicate which TF families cooperate with and antagonize each other to regulate adipogenesis. Compartment modeling of RNA polymerase density quantifies how individual TFs mechanistically contribute to distinct steps in transcription. Glucocorticoid receptor activates transcription by inducing RNA polymerase pause release while SP and AP1 factors affect RNA polymerase initiation. We identify Twist2 as a previously unappreciated effector of adipocyte differentiation. We find that TWIST2 acts as a negative regulator of 3T3-L1 and primary preadipocyte differentiation. We confirm that Twist2 knockout mice have compromised lipid storage within subcutaneous and brown adipose tissue. Previous phenotyping of Twist2 knockout mice and Setleis syndrome (Twist2-/-) patients noted deficiencies in subcutaneous adipose tissue. This network inference framework is a powerful and general approach for interpreting complex biological phenomena and can be applied to a wide range of cellular processes.

Heat activation and inactivation of bacterial spores. Is there an overlap?

Wen, J. — 2021-11-20

Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore germination. This treatment also has potential to be employed in food processing to eliminate undesired bacterial spores by enhancing their germination, and then inactivating the less heat resistant germinated spores at a milder temperature. However, incorrect heat treatment could also generate heat damage in spores, and lead to more heterogeneous spore germination. Here, the heat activation and heat damage profile of Bacillus subtilis spores was determined by testing spore germination and outgrowth at both population and single spore levels. The heat treatments used were 40-80{degrees}C, and for 0-300 min. The results were as follows. 1) Heat activation at 40-70{degrees}C promoted L-valine and L-asparagine-glucose-fructose-potassium (AGFK) induced germination in a time dependent manner. 2) The optimal heat activation temperatures for AGFK and L-valine germination via the GerB plus GerK or GerA germinant receptors were 65 and 50-65{degrees}C, respectively. 3) Heat inactivation of dormant spores appeared at 70{degrees}C, and the heat damage of molecules essential for germination and growth began at 70 and 65{degrees}C, respectively. 4) Heat treatment at 75{degrees}C resulted in both activation of germination and damage to the germination apparatus, and 80{degrees}C treatment caused more pronounced heat damage. 5) For the spores that should withstand adverse environmental temperatures in nature, heat activation seems functional for a subsequent optimal germination process, while heat damage affected both germination and outgrowth.

Organization and dynamics of the SpoVAEa protein, and its surrounding inner membrane lipids upon germination of Bacillus subtilis spores

Wen, J. — 2021-11-20

The SpoVA proteins make up a channel in the inner membrane (IM) of B. subtilis spore. This channel responds to signals from activated germinant receptors (GRs), and allows release of Ca2+-DPA from the spore core during germination. In the current work, we studied the location and dynamics of SpoVAEa in dormant spores. Notably, the SpoVAEa-SGFP2 proteins were present in a single spot in spores, similar to the complex formed by all GRs. However, while the GRs spot remains in one location, the SpoVAEa-SGFP2 spot in the IM moved randomly with high frequency. The dynamics of the SpoVAEa-SGFP2 and its surrounding IM region as stained by fluorescent dyes were also tracked during spore germination, as the dormant spore IM appeared to have an immobile germination related functional microdomain. This microdomain disappeared around the time of appearance of a germinated spore, the loss of fluorescence of the IM by fluorescent dyes, as well as the appearance of SpoVAEa-SGFP2 peak fluorescent intensity occurred in parallel. These observed events were highly related to the rapid phase darkening, which is considered as the Ca2+DPA rapid release. We also tested the response of SpoVAEa and the IM to thermal treatments at 40-80{degrees}C. Heat treatment triggered an increase of green autofluorescence, which is speculated to be due to coat protein denaturation, and 80{degrees}C treatments induce the appearance of phase-grey-like spores. These spores presumably have a similar intracellular physical state as the phase grey spores detected in the germination but lack the functional proteins for further germination events.

Monosomy X in isogenic human iPSC-derived trophoblast model impacts expression modules preserved in human placenta

Ahern, D. T. — 2021-12-14

SUMMARY/ABSTRACTMammalian sex chromosomes encode homologous X/Y gene pairs that were retained on the male Y and escape X chromosome inactivation (XCI) in females. Inferred to reflect X/Y-pair dosage sensitivity, monosomy X is a leading cause of miscarriage in humans with near full penetrance. This phenotype is shared with many other mammals but not the mouse, which offers sophisticated genetic tools to generate sex chromosomal aneuploidy but also tolerates its developmental impact. To address this critical gap, we generated X-monosomic human induced pluripotent stem cells (hiPSCs) alongside otherwise isogenic euploid controls from male and female mosaic samples. Phased genomic variants of these hiPSC panels enable systematic investigation of X/Y dosage-sensitive features using in vitro models of human development. Here, we demonstrate the utility of these validated hiPSC lines to test how X/Y-linked gene dosage impacts a widely-used model for the human syncytiotrophoblast. While these isogenic panels trigger a GATA2/3 and TFAP2A/C -driven trophoblast gene circuit irrespective of karyotype, differential expression implicates monosomy X in altered levels of placental genes, and in secretion of placental growth factor (PlGF) and human chorionic gonadotropin (hCG). Remarkably, weighted gene co-expression network modules that significantly reflect these changes are also preserved in first-trimester chorionic villi and term placenta. Our results suggest monosomy X may skew trophoblast cell type composition, and that the pseudoautosomal region likely plays a key role in these changes, which may facilitate prioritization of haploinsufficient drivers of 45,X extra-embryonic phenotypes.

A calibrated cell-based functional assay to aide classification of MLH1 DNA mismatch repair gene variants

Rath, A. — 2021-12-14

PURPOSEFunctional assays provide important evidence for classifying the disease significance of germline variants in the DNA mismatch repair genes. We sought to develop a cell-based approach for testing the function of variants of uncertain significance (VUS) in the MLH1 gene. METHODSUsing CRISPR gene editing, we knocked-in MLH1 VUS into the endogenous MLH1 loci in human embryonic stem cells. We examined their impact at the RNA and protein level, including their ability to maintain stability of microsatellite sequences and instigate a DNA damage response. We calibrated these assays by testing well-established pathogenic and benign control variants. RESULTSFive VUS resulted in functionally abnormal protein, 15 VUS resulted in functionally normal protein, and one VUS showed mixed results. Furthermore, we converted the functional outputs into a single odds in favor of pathogenicity score for each VUS. CONCLUSIONOur CRISPR-based functional assay successfully models phenotypes observed in patients in a cellular context. Using this approach, we generated evidence for or against pathogenicity for utilization by variant classification expert panels. Ultimately, this information will assist in proper diagnosis and disease management for suspected Lynch syndrome patients.

A reference induced pluripotent stem cell line for large-scale collaborative studies

Pantazis, C. B. — 2021-12-17

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate iPSC lines and deeply characterised their genetic properties using whole genome sequencing, their genomic stability upon CRISPR/Cas9-based gene editing, and their phenotypic properties including differentiation to commonly-used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and hundreds of its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field. SummaryThe authors of this collaborative study deeply characterized human induced pluripotent stem cell (iPSC) lines to rationally select a clonally-derived cell line that performs well across multiple modalities. KOLF2.1J was identified as a candidate reference cell line based on single-cell analysis of its gene expression in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and the efficiency with which it differentiated into multiple target cell populations. Since it is deeply characterized and can be readily acquired, KOLF2.1J is an attractive reference cell line for groups working with iPSCs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/472643v6_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@cef106org.highwire.dtl.DTLVardef@31d5d8org.highwire.dtl.DTLVardef@1ce6d8corg.highwire.dtl.DTLVardef@17a19d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

SMAD4 and TGFβ are architects of inverse genetic programs during fate-determination of antiviral memory CD8 T cells

Chandiran, K. — 2021-12-16

Transforming growth factor {beta} (TGF{beta}) is a morphogenic protein that augments antiviral immunity by altering the functional properties of pathogen-specific memory CD8 T cells. During infection TGF{beta} inhibits formation of effector (TEFF) and central memory CD8 T cells (TCM), while encouraging tissue-resident memory CD8 T cells (TRM) to settle in peripheral tissues. SMAD proteins are signaling intermediates that are used by members of the TGF cytokine family to modify gene expression. For this study, RNA-sequencing was used to explore how regulation via SMAD4 alters the transcriptional profile of antiviral CTLs during infection. We show that SMAD4 and TGF{beta} cooperatively regulate a collection of genes that determine whether specialized populations of pathogen-specific CTLs circulate around the body, or settle in peripheral tissue. The target genes include multiple homing receptors (CD103, KLRG1 and CD62L) and transcription factors (Hobit and EOMES) that support memory formation. While TGF{beta} uses a canonical SMAD-dependent signaling pathway to induce CD103 expression on TRM cells, an alternative SMAD4-dependent mechanism is required for formation of TEFF and TCM cells in the circulation. Graphical abstractTGF{beta} and SMAD4 modulate gene expression in reciprocal directions during differentiation of antiviral CTLs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/472993v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@5ac140org.highwire.dtl.DTLVardef@455a2aorg.highwire.dtl.DTLVardef@1227d96org.highwire.dtl.DTLVardef@16336b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Membrane voltage fluctuations in human breast cancer cells

Quicke, P. — 2021-12-21

Cancer cells feature a resting membrane potential (Vm) that is depolarized compared to normal cells, and express active ionic conductances, which factor directly in their pathophysiological behavior. Despite similarities to excitable tissues, relatively little is known about cancer cell Vm dynamics. With high-throughput, cellular-resolution Vm imaging, we characterized Vm fluctuations of hundreds of human triple-negative breast cancer MDA-MB-231 cells and compared to non-cancerous breast epithelial MCF-10A cells. By quantifying their Dynamic Electrical Signatures (DESs) through an unsupervised machine-learning protocol, we identified four classes ranging from "noisy" to "blinking/waving". The Vm of MDA-MB-231 cells exhibited spontaneous, transient hyperpolarizations that were inhibited by the voltage-gated sodium channel blocker tetrodotoxin. The Vm of MCF-10A cells was comparatively static, but fluctuations increased following treatment with transforming growth factor-{beta}1, a canonical inducer of the epithelial-to-mesenchymal transition. These data suggest that the ability to generate Vm fluctuations is acquired during transformation and may participate in oncogenesis.

NETISCE: A Network-Based Tool for Cell Fate Reprogramming

Marazzi, L. — 2022-01-01

The search for effective therapeutic targets in fields like regenerative medicine and cancer research has generated interest in cell fate reprogramming. This cellular reprogramming paradigm can drive cells to a desired target state from any initial state. However, methods for identifying reprogramming targets remain limited for biological systems that lack large sets of experimental data or a dynamical characterization. We present NETISCE, a novel computational tool for identifying cell fate reprogramming targets in static networks. In combination with machine learning algorithms, NETISCE estimates the attractor landscape and predicts reprogramming targets using Signal Flow Analysis and Feedback Vertex Set Control, respectively. Through validations in studies of cell fate reprogramming from developmental, stem cell, and cancer biology, we show that NETISCE can predict previously identified cell fate reprogramming targets and identify potentially novel combinations of targets. NETISCE extends cell fate reprogramming studies to larger-scale biological networks without the need for full model parameterization and can be implemented by experimental and computational biologists to identify parts of a biological system relevant to the desired reprogramming task.

Computational modeling of macrophage iron sequestration during host defense against Aspergillus

Adhikari, B. — 2022-01-27

Iron is essential to the virulence of Aspergillus species, and restricting iron availability is a critical mechanism of antimicrobial host defense. Macrophages recruited to the site of infection are at the crux of this process, employing multiple intersecting mechanisms to orchestrate iron sequestration from pathogens. To gain an integrated understanding of how this is achieved in invasive aspergillosis, we generated a transcriptomic time-series of the response of human monocyte-derived macrophages to Aspergillus and used this and the available literature to construct a mechanistic computational model of iron handling of macrophages during this infection. We found an overwhelming macrophage response beginning 2-4 hours after exposure to the fungus, which included upregulated transcription of iron import proteins transferrin receptor-1, divalent metal transporter-1, and ZIP family transporters, and downregulated transcription of the iron exporter ferroportin. The computational model, based on a discrete dynamical systems framework, consisted of 21 3-state nodes, and was validated with additional experimental data that were not used in model generation. The model accurately captures the steady state and the trajectories of most of the quantitatively measured nodes. In the experimental data, we surprisingly found that transferrin receptor-1 upregulation preceded the induction of inflammatory cytokines, a feature that deviated from model predictions. Model simulations suggested that direct induction of TfR1 after fungal recognition, independent of the Iron Regulatory Protein - Labile Iron Pool system, explains this finding. We anticipate that this model will contribute to a quantitative understanding of iron regulation as a fundamental host defense mechanism during aspergillosis. ImportanceInvasive pulmonary aspergillosis is a major cause of death among immunosuppressed individuals despite the best available therapy. Depriving the pathogen of iron is an essential component of host defense in this infection, but the mechanisms by which the host achieves this are complex. To understand how recruited macrophages mediate iron deprivation during the infection, we developed and validated a mechanistic computational model that integrates the available information in the field. The insights provided by this approach can help in designing iron modulation therapies as anti-fungal treatments.

Integrative analysis of transcriptomics in human craniofacial development reveals novel candidate disease genes

Yankee, T. N. — 2022-02-28

Craniofacial disorders are among the most common of all congenital defects. A majority of craniofacial development occurs early in pregnancy and to fully understand how craniofacial defects arise, it is essential to observe gene expression during this critical time period. To address this we performed bulk and single-cell RNA-seq on human craniofacial tissue from embryonic development 4 to 8 weeks post conception. This data comprises the most comprehensive profiling of the transcriptome in the early developing human face to date. We identified 239 genes that were specifically expressed in craniofacial tissues relative to dozens of other human tissues and stages. We found that craniofacial specific enhancers are enriched within 400kb of these genes establishing putative regulatory interactions. To further understand how genes are organized in this program we constructed coexpression networks. Strong disease candidates are likely genes that are coexpressed with many other genes, serving as regulatory hubs within these networks. We leveraged large functional genomics databases including GTEx and GnomAD to reveal hub genes that are specifically expressed in craniofacial tissue and genes which are resistant to mutation in the normal healthy population. Our unbiased method revealed dozens of novel disease candidate genes that warrant further study.

Phospholipase Cβ2 Promotes Vascular Endothelial Growth Factor Induced Vascular Permeability

Phoenix, K. N. — 2022-03-10

BackgroundRegulation of vascular permeability (VP) is critical to maintaining tissue metabolic homeostasis. Vascular endothelial growth factor (VEGF) is a key stimulus of VP in acute and chronic diseases including ischemia reperfusion injury, sepsis and cancer. Identification of novel regulators of VP would allow for the development of effective targeted therapeutics for patients with unmet medical need. MethodsIn vitro and in vivo models of VEGFA-induced vascular permeability, pathological permeability, quantitation of intracellular calcium release and cell entry, and PIP2 levels were evaluated with and without modulation of PLC{beta}2. ResultsGlobal knock-out of PLC{beta}2 in mice resulted in blockade of VEGFA-induced vascular permeability in vivo and trans-endothelial permeability in primary lung endothelial cells. Further work in an immortalized human microvascular cell line modulated with stable knock-down of PLC{beta}2 recapitulated the observations in the mouse model and primary cell assays. Additionally, loss of PLC{beta}2 limited both intracellular release and extracellular entry of calcium following VEGF stimulation as well as reduced basal and VEGFA-stimulated levels of PIP2 compared to control cells. Finally, loss of PLC{beta}2 in both a hyperoxia induced lung permeability model and a cardiac ischemia:reperfusion model resulted in improved animal outcomes when compared to WT controls. ConclusionsThe results implicate PLC{beta}2 as a key positive regulator of VEGF-induced VP through regulation of both calcium flux and PIP2 levels at the cellular level. Targeting of PLC{beta}2 in a therapeutic setting may provide a novel approach to regulating vascular permeability in patients. Graphic Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY HighlightsO_LIPLC{beta}2 promotes VEGFA induced vascular permeability. C_LIO_LILoss of PLC{beta}2 prevents VEGFA vascular permeability via repression of cellular calcium flux and membrane PIP2 levels. C_LIO_LILoss of PLC{beta}2 reduces vascular permeability and improves outcomes in a hyperoxic lung damage model and a cardiac ischemia:reperfusion model in vivo. C_LIO_LITargeting PLC{beta}2 inhibition may lead to a novel therapeutic for diseases such as stroke and myocardial infarction. C_LI

A distant global control region is essential for normal expression of anterior HOXA genes during mouse and human craniofacial development.

Wilderman, A. — 2022-03-11

Defects in embryonic patterning resulting in craniofacial abnormalities account for approximately 1/3 of birth defects. The regulatory programs that build and shape the face require precisely controlled spatiotemporal gene expression, achieved through tissue-specific enhancers. Large regions with coactivation of enhancer elements and co-regulation of multiple genes, referred to as superenhancers, are important in determining cell identity and perturbation could result in developmental defects. Building upon a previously published epigenomic atlas of human embryonic craniofacial tissue in which we identified over 75,000 putative embryonic craniofacial enhancer regions, we have identified 531 superenhancer regions unique to embryonic craniofacial tissue, including 37 which fall in completely noncoding regions. To demonstrate the utility of this data for the understanding of craniofacial development and the etiology of craniofacial abnormalities, we focused on a craniofacial-specific superenhancer in a [~]600kb noncoding region located between NPVF and NFE2L3. This region harbors over 100 individual putative craniofacial enhancer segments and 7 in vivo validated craniofacial enhancers from primary craniofacial tissue as well as strong enhancer activation signatures in a culture model of cranial neural crest cell (CNCC) development. However, none of the directly adjacent genes have been implicated in neural crest specification, craniofacial development, or abnormalities. To identify potential regulatory targets of this superenhancer region, we characterized three-dimensional chromatin structure of this region in CNCCs and mouse embryonic craniofacial tissues using multiple techniques (4C-Seq, HiC). We identified long range interactions that exclude most intervening genes and specifically target the anterior portion of the HOXA gene cluster located 1.2 to 1.8 Mb away. We demonstrate the specificity of the enhancer region for regulation of anterior HOXA genes through CRISPR/Cas9 editing of human embryonic stem cells. Mice homozygous for deletion of the superenhancer confirm the specificity of the enhancer region and demonstrate that the region is essential for viability. At fetal stages homozygotes develop at the same rate as heterozygous and wild type littermates but die at P0-P1 and have high penetrance of orofacial clefts that phenocopy previously described Hoxa2-/- mice. Moreover, we identified a de novo deletion partially overlapping the superenhancer in a human fetus with severe craniofacial abnormalities. This evidence suggests we have identified a critical noncoding locus control region that specifically regulates anterior HOXA genes and whose deletion is likely pathogenic in human patients.

The role of UBE3A in the autism and epilepsy-related Dup15q syndrome using patient-derived, CRISPR-corrected neurons

Elamin, M. — 2022-03-12

Chromosome 15q11-q13 duplication syndrome (Dup15q) is a neurodevelopmental disorder caused by maternal duplications of this region. Autism and epilepsy are key features of Dup15q, but affected individuals also exhibit intellectual disability and developmental delay. UBE3A, the gene encoding the ubiquitin protein ligase E3A, is likely a major driver of Dup15q because individuals with maternal, but not paternal 15q duplications have the disorder, and UBE3A is the only imprinted gene expressed solely from the maternal allele in mature neurons. Nevertheless, the exact role of UBE3A is yet to be determined. To establish whether UBE3A overexpression is required for Dup15q neuronal deficits, UBE3A expression was manipulated in patient-derived induced pluripotent stem cell (iPSC) lines using antisense oligonucleotides. Dup15q neurons exhibited hyperexcitability features compared to genome-edited isogenic control neurons, and this phenotype was generally prevented by normalizing UBE3A levels throughout in vitro development. Overexpression of UBE3A in an iPSC line with a paternal duplication resulted in a profile similar to that of Dup15q neurons except for synaptic phenotypes. These results indicate that UBE3A overexpression is necessary for most Dup15q cellular phenotypes. However, the inability of UBE3A overexpression to recapitulate synaptic phenotypes suggests an important role for non-imprinted genes in the disorder.

Integration of multimodal data in the developing tooth reveals candidate dental disease genes

Winchester, E. W. — 2022-03-16

Dental malformations range from rare syndromes to common nonsyndromic phenotypes. These malformations can predispose individuals to dental disease, which can in turn affect systemic health. While many dental phenotypes are heritable, most cases have not been linked to deleterious mutations in single genes. We demonstrate that human and conserved mouse craniofacial enhancers show enrichment of dental phenotype-associated variants. Given these findings in bulk craniofacial tissues, we looked to determine the role of tooth enhancers in this phenomenon. We used ChIP-seq and machine learning to identify enhancers of E13.5 mouse incisors. Multi-tissue comparisons of human and mouse enhancers revealed that putative tooth enhancers had the strongest enrichment of dental phenotype-associated variants, suggesting a role for dysregulation of tooth development in dental phenotypes. To uncover novel dental phenotype-driving genes in the developing tooth we performed coexpression analysis and annotated the contributing cell types of gene modules using scRNAseq. Through integration of chromatin state, bulk gene coexpression, and cell type resolved gene expression we prioritized a list of candidate novel dental disease genes for future investigations in mouse models and human studies.

Modeling of Aryl Hydrocarbon Receptor Pathway Intrinsic Immunometabolic Role using Glioblastoma Stem Cells and Patient-Derived Organoids

Watanabe, F. — 2022-03-19

The intrinsic genetic program of glioblastoma (GBM) stem cells is critical for tumor evolution and recurrence. We recently identified intrinsic phenotypes and immune-like genetic programs of GBM organoids (GBMO)1 from patient derived glioblastoma stem cells (GSCs), replicating genomic, metabolic, and cellular aspects of GBM in vivo. Aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, is a key regulator of infiltrating immune cells in gliomas2, 3 and associated with poor prognosis, but its role in GSC biology is unknown2. Here, we show that AHR is a patient-specific regulator of the glioma intrinsic gene program in GSCs and GSC-derived GBMO that are enriched for AHR. We find that AHR is required for GSC self-renewal, GBMO expansion, radial glia-like cell proliferation, and expression of immune mediators seen in the mesenchymal subtype. CRISPR-Cas9 genetic ablation and pharmacological inhibition revealed that AHR regulates genes linked to intrinsic immunity, proliferation, and migration in GBMO. Genomic analysis of GBMO treated with AHR inhibitors identified expression signatures and candidate markers associated with survival of gliomas. Our work defines the glioma intrinsic function of AHR in a model of early GBM formation, offering a rationale for clinical exploration of a potential two-hit target of both GBM cells and infiltrating immune cells in patients with GBM expressing high levels of AHR.

Endogenous production of hyaluronan, PRG4, and cytokines is sensitive to cyclic loading in synoviocytes

Pendyala, M. — 2022-04-20

Synovial fluid is composed of hyaluronan and proteoglycan-4 (PRG4 or lubricin), which work synergistically to maintain joint lubrication. In diseases like osteoarthritis, hyaluronan and PRG4 concentrations can be altered, resulting in lowered synovial fluid viscosity, and pro-inflammatory cytokine concentrations within the synovial fluid increase. Synovial fibroblasts within the synovium are responsible for contributing to synovial fluid and can be targeted to improve endogenous production of hyaluronan and PRG4 and to alter the cytokine profile. We cyclically loaded SW982 synoviocytes to 0%, 5%, 10%, or 20% strain for three hours at 1 Hz. To assess the impact of substrate stiffness, we compared the 0% strain group to cells grown on tissue culture plastic. We measured the expression of hyaluronan turnover genes, hyaluronan localization within the cell layer, hyaluronan concentration, PRG4 concentration, and the cytokine profile within the media. Our results show that the addition of cyclic loading increased HAS3 expression, but not in a magnitude-dependent response. Hyaluronidase expression was impacted by strain magnitude, which is exemplified by the decrease in hyaluronan concentration due to cyclic loading. We also show that PRG4 concentration is increased at 5% strain, while higher strain magnitude decreases overall PRG4 concentration. Finally, 10% and 20% strain show a distinct, more pro-inflammatory cytokine profile when compared to the unloaded group. Multivariate analysis showed distinct separation between certain strain groups in being able to predict strain group, hyaluronan concentration, and PRG4 concentration from gene expression or cytokine concentration data, highlighting the complexity of the system. Overall, this study shows that cyclic loading can be used tool to modulate the endogenous production of hyaluronan, PRG4, and cytokines from synovial fibroblasts.

A dynamic in vitro model of Down Syndrome neurogenesis with Trisomy 21 gene dosage correction

Bansal, P. — 2022-05-11

ABSTRACT/SUMMARYExcess gene dosage from human chromosome 21 (chr21) causes Down syndrome (DS), spanning developmental as well as acute phenotypes in terminal cell types. Which phenotypes remain amenable to intervention after development is unknown. To address this question in a model of DS neurogenesis, we generated trisomy 21 (T21) human induced pluripotent stem cells (hiPSCs) alongside otherwise isogenic euploid controls from mosaic DS fibroblasts, and integrated an inducible XIST transgene on one chr21 copy. Monoallelic chr21 silencing by XIST was near-complete and irreversible in hiPSCs. Differential expression reveals T21 neural lineages and T21 hiPSCs suppress similar translation and mitochondrial pathways, and activate cellular stress responses. When XIST is induced before the neural progenitor stage, T21 dosage correction mitigates a pronounced skew towards astrogenesis in differentiation. Because our transgene remained inducible in post-mitotic T21 neurons and astrocytes, we demonstrate XIST efficiently represses genes even after terminal differentiation, which will empower exploration of cell type-specific T21 phenotypes that remain responsive to chr21 dosage.

Channel-independent function of UNC-9/INX in spatial arrangement of GABAergic synapses in C. elegans

Hendi, A. — 2022-05-16

Precise synaptic connection of neurons with their targets is essential for the proper functioning of the nervous system. A plethora of signaling pathways act in concert to mediate the precise spatial arrangement of synaptic connections. Here we show a novel role for a gap junction protein in controlling tiled synaptic arrangement in the GABAergic motor neurons in C. elegans, in which their axons and synapses overlap minimally with their neighboring neurons within the same class. We found that while EGL-20/Wnt controls axonal tiling, their presynaptic tiling is mediated by a gap junction protein UNC-9/Innexin, that is localized at the presynaptic tiling border between neighboring DD neurons. Strikingly, the gap junction channel activity of UNC-9 is dispensable for its function in controlling tiled presynaptic patterning. While gap junctions are crucial for the proper functioning of the nervous system as channels, our finding uncovered the novel channel-independent role of UNC-9 in synapse patterning.

Efficient End-to-end Learning for Cell Segmentation with Machine Generated Incomplete Annotations

Shrestha, P. — 2022-07-03

Automated cell segmentation from optical microscopy images is usually the first step in the pipeline of single-cell analysis. Recently, deep-learning based algorithms have shown superior performances for the cell segmentation tasks. However, a disadvantage of deep-learning is the requirement for a large amount of fully-annotated training data, which is costly to generate. Weakly-supervised and self-supervised learning is an active research area, but often the model accuracy is inversely correlated with the amount of annotation information provided. Here we focus on a specific subtype of incomplete annotations, which can be generated programmably from experimental data, thus allowing for more annotation information content without sacrificing the annotation speed. We designed a new model architecture for end-to-end training using such incomplete annotations. We benchmarked our method on a variety of publicly available dataset, covering both fluorescence and bright-field imaging modality. We additionally tested our method on a microscopy dataset generated by us, using machine generated annotations. The results demonstrated that our model trained under weak-supervision can achieve segmentation accuracy competitive to, and in some cases surpassing, state-of-the-art models trained under full supervision. Therefore, our method can be a practical alternative to the established full-supervision methods.

Monoallelically-expressed Noncoding RNAs form nucleolar territories on NOR-containing chromosomes and regulate rRNA expression

Hao, Q. — 2022-07-04

Out of the several hundred copies of rRNA genes that are arranged in the nucleolar organizing regions (NOR) of the five human acrocentric chromosomes, [~]50% remain transcriptionally inactive. NOR-associated sequences and epigenetic modifications contribute to differential expression of rRNAs. However, the mechanism(s), controlling the dosage of active versus inactive rRNA genes in mammals is yet to be determined. We have discovered a family of ncRNAs, SNULs (Single NUcleolus Localized RNA), which form constrained sub-nucleolar territories on individual NORs and influences rRNA expression. Individual members of the SNULs monoallelically associate with specific NOR-containing chromosome. SNULs share sequence similarity to pre-rRNA and localize in the sub-nucleolar compartment with pre-rRNA. Finally, SNULs control rRNA expression by influencing pre-rRNA sorting to the DFC compartment and pre-rRNA processing. Our study discovered a novel class of ncRNAs that by forming constrained nucleolar territories on individual NORs contribute to rRNA expression.

Approximating Scaffold Printability Utilizing Computational Methods

Sedigh, A. — 2022-07-11

Bioprinting facilitates the generation of complex, three-dimensional (3D), cell-based constructs for various applications. Although multiple bioprinting technologies have been developed, extrusion-based systems have become the dominant technology due to the diversity of materials (bioinks) that can be utilized, either individually or in combination. However, each bioink has unique material properties and extrusion characteristics that affect bioprinting utility, accuracy, and precision. Here, we have extended our previous work to achieve high precision (i.e., repeatability) across samples by optimizing bioink-specific printing parameters. Specifically, we hypothesized that an adaptive neuro-fuzzy inference system (ANFIS) could be used as a computational method to address the imprecision in 3D bioprinting test data and uncover the optimal printing parameters for a specific bioink that result in high accuracy and precision. To test this hypothesis, we have implemented an ANFIS model consisting of four inputs (bioink concentration, printing pressure, speed, and temperature) and a single output to quantify the precision (scaffold bioprinted linewidth range). We validate our use of the bioprinting precision index (BPI) with both standard and normalized printability factors. In total, our results indicate that computational methods are a cost-efficient measure to improve the precision and robustness of extrusion 3D bioprinting with gelatin-based bioinks.

MIADE metadata guidelines: Minimum Information About a Disorder Experiment

Meszaros, B. — 2022-07-14

An unambiguous description of an experimental setup and analysis, and the subsequent biological observation is vital for accurate data interpretation and reproducible results. Consequently, experimental analyses should be described in a concise, unequivocal, and digestible manner. The aim of minimum information guidelines is to define the fundamental complement of data that can support an unambiguous conclusion on experimental observations. In this document, we present the Minimum Information About Disorder Experiments (MIADE) guidelines to define the minimal fundamental parameters required for non-experts to understand the key findings of an experiment studying intrinsically disordered proteins (IDPs) or intrinsically disordered protein regions (IDRs). MIADE guidelines provide recommendations for data producers to describe the results of their experiments at source, for curators to annotate experimental data to community resources and for database developers maintaining community resources to disseminate the data. We give examples of the application of these guidelines in common use cases and describe the implementation of an update to the DisProt IDP database to allow MIADE-compliant annotation. The MIADE guidelines will improve the interpretability of experimental results for data consumers, facilitate direct data submission, simplify data curation, improve data exchange among repositories and standardise the dissemination of the key metadata on an IDP experiment by IDP data sources.

High-throughput nanopore sequencing of Treponema pallidum tandem repeat genes arp and tp0470 reveals clade-specific patterns and recapitulates global whole genome phylogeny

Lieberman, N. A. P. — 2022-08-02

Sequencing of most Treponema pallidum (T. pallidum) genomes excludes repeat regions in tp0470 and the tp0433 gene, encoding the acidic repeat protein (arp). As a first step to understanding the evolution and function of these genes and the proteins they encode, we developed a protocol to nanopore sequence tp0470 and arp genes from 212 clinical samples collected from ten countries on six continents. Both tp0470 and arp repeat structures recapitulate the whole genome phylogeny, with subclade-specific patterns emerging. The number of tp0470 repeats is on average appears to be higher in Nichols-like clade strains than in SS14-like clade strains. Consistent with previous studies, we found that 14-repeat arp sequences predominate across both major clades, but the combination and order of repeat type varies among subclades, with many arp sequence variants limited to a single subclade. Although strains that were closely related by whole genome sequencing frequently had the same arp repeat length, this was not always the case. Structural modelling of TP0470 suggested that the eight residue repeats form an extended -helix, predicted to be periplasmic. Modeling of the ARP revealed a C-terminal sporulation-related repeat (SPOR) domain, predicted to bind denuded peptidoglycan, with repeat regions possibly incorporated into a highly charged {beta}- sheet. Outside of the repeats, all TP0470 and ARP amino acid sequences were identical. Together, our data, along with functional considerations, suggests that both TP0470 and ARP proteins may be involved in T. pallidum cell envelope remodeling and homeostasis, with their highly plastic repeat regions playing as-yet-undetermined roles.

Non-canonical Telomerase Reverse Transcriptase Controls Osteogenic Differentiation of Aortic Valve Cells Through STAT5

Cuevas, R. A. — 2022-08-23

BackgroundCalcific aortic valve disease (CAVD) is the pathological remodeling of valve leaflets. The initial steps in valve leaflet osteogenic reprogramming are not fully understood. As telomerase reverse transcriptase (TERT) overexpression primes mesenchymal stem cells to differentiate into osteoblasts, we investigated whether TERT contributes to the osteogenic reprogramming of valve interstitial cells (VICs). MethodsHuman control and CAVD aortic valve leaflets and patient-specific hVICs were used in in vivo and in vitro calcification assays. Loss of function experiments in hVICs and cells isolated from Tert-/-and Terc-/- mice were used for mechanistic studies. Calcification was assessed in Tert+/+ and Tert-/- mice ex vivo and in vivo. In silico modeling, proximity ligation and co-immunoprecipitation assays defined novel TERT interacting partners. Chromatin immunoprecipitation and CUT&TAG sequencing defined protein-DNA interactions. ResultsTERT protein was highly expressed in calcified valve leaflets without changes in telomere length, DNA damage, or senescence markers, and these features were retained in isolated primary hVICs. TERT expression increased with osteogenic or inflammatory stimuli, and knock-down or genetic deletion of TERT prevented calcification in vitro and in vivo. Mechanistically, TERT was upregulated via NF-{kappa}B and required to initiate osteogenic reprogramming, independent of its canonical reverse transcriptase activity and the lncRNA TERC. TERT exerts non-canonical osteogenic functions via binding with Signal Transducer and Activator of Transcription 5 (STAT5). Depletion or inhibition of STAT5 prevented calcification. STAT5 was found to bind the promoter region of Runt-Related Transcription Factor 2 (RUNX2), the master regulator of osteogenic reprogramming. Lastly, we demonstrate that TERT and STAT5 are upregulated and colocalized in CAVD tissue compared to control tissue. ConclusionsTERTs non-canonical activity is required to initiate calcification. TERT is upregulated via inflammatory signaling pathways and partners with STAT5 to bind the RUNX2 gene promoter. These data identify a novel mechanism and potential therapeutic target to decrease vascular calcification. Novelty and SignificanceWhat is known? Calcific aortic valve disease (CAVD) is the most prevalent form of aortic valve pathology. CAVD strongly correlates with age and leads to heart failure and a high risk of stroke. Currently, the only therapeutic option is valve replacement, which comes with significant healthcare costs and additional risks to patients. Runt-related transcription factor 2 (RUNX2) is the master transcription factor required for osteogenic differentiation of stem cells to osteoblasts and osteogenic reprogramming of cardiovascular cells. Yet, the early events driving its activity in aortic valve cells are poorly defined. In addition to its reverse transcriptase enzymatic activity, TERT exhibits non-canonical transcriptional regulatory functions and overexpression of TERT primes mesenchymal stem cells to differentiate down the osteoblast lineage. What new information does this article contribute? TERT protein levels in calcified aortic leaflets and valve interstitial cells, and its non-canonical osteogenic activity are independent of changes in telomere length and cell senescence. Genetic loss or depletion of TERT prevented calcification in valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells in vitro and the vasculature in vivo. Early in the osteogenic reprogramming inflammatory signaling promotes TERT to co-localize with SMARCA4 and STAT5, and this TERT-tethered STAT5 binds to the RUNX2 gene promoter, the master regulator of osteogenic transcriptional programs. STAT5 depletion and pharmacological inhibition prevent calcification of human valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells. What are the clinical implications? We have identified TERT-STAT5 as a novel signaling axis that orchestrates the early steps in the osteogenic reprogramming of aortic valve cells. Inhibiting TERT/STAT5 interaction or their activity may be leveraged for the development of therapeutic strategies to halt or prevent calcification in the aortic valve, bioprosthetic valves, and or perhaps other cardiovascular tissues. Invasive and expensive surgical procedures are currently the only treatment option for patients with CAVD. The discovery and defining of the early events driving vascular calcification identifies novel and druggable targets for developing non-surgical therapies.

Microbial glutamate metabolism predicts intravenous cocaine self-administration in Diversity Outbred mice

Tran, T. D. B. — 2022-09-13

The gut microbiome is thought to play a critical role in the onset and development of psychiatric disorders, including depression and substance use disorder (SUD). To test the hypothesis that the microbiome affects addiction predisposing behaviors and cocaine intravenous self-administration (IVSA) and to identify specific microbes involved in the relationship, we performed 16S rRNA gene sequencing on feces from 228 diversity outbred mice. Twelve open field measures, two light-dark assay measures, one hole board and novelty place preference measure significantly differed between mice that acquired cocaine IVSA (ACQ) and those that failed to acquire IVSA (FACQ). We found that ACQ mice are more active and exploratory and display decreased fear than FACQ mice. The microbial abundances that differentiated ACQ from FACQ mice were an increased abundance of Barnesiella, Ruminococcus, and Robinsoniella and decreased Clostridium IV in ACQ mice. There was a sex-specific correlation between ACQ and microbial abundance, a reduced Lactobacillus abundance in ACQ male mice, and a decreased Blautia abundance in female ACQ mice. The abundance of Robinsoniella was correlated, and Clostridium IV inversely correlated with the number of doses of cocaine self-administered during acquisition. Functional analysis of the microbiome composition of a subset of mice suggested that gut-brain modules encoding glutamate metabolism genes are associated with the propensity to self-administer cocaine. These findings establish associations between the microbiome composition and glutamate metabolic potential and the ability to acquire cocaine IVSA thus indicating the potential translational impact of targeting the gut microbiome or microbial metabolites for treatment of SUD. HIGHLIGHTSO_LICorrelational analysis of novelty behaviors to IVSA acquisition shows that mice that acquire cocaine IVSA are more active and exploratory and have decreased fear than those that failed-to-acquire IVSA. C_LIO_LIThe gut microbiome profiling of 228 diversity outbred mice indicates the relative abundances of Barnesiella, Ruminococcus, Robinsoniella and Clostridium IV are associated with the ability to self-administer cocaine. C_LIO_LIAssociations between the gut microbiome and IVSA acquisition are sex-specific. Decreased relative abundances of Lactobacillus and Blautia are associated with IVSA in male and female mice, respectively. C_LIO_LIThe relative abundances of Robinsoniella and Clostridium IV were correlated with the number of infusions of self-administered cocaine. C_LIO_LIFunctional potential analysis of the gut microbiome supports a role for microbiomes encoding glutamate metabolism in the ability to self-administer cocaine. C_LI

PhysiPKPD: A pharmacokinetics and pharmacodynamics module for PhysiCell

Bergman, D. R. — 2022-09-15

Pharmacokinetics and pharmacodynamics are key considerations in any study of molecular therapies. It is thus imperative to factor their effects in to any in silico model of biological tissue involving such therapies. Furthermore, creation of a standardized and flexible framework will benefit the community by increasing access to such modules and enhancing their communicability. PhysiCell is an open source physics-based cell simulator, i.e. a platform for modeling biological tissue, that is quickly being adopted and utilized by the mathematical biology community. We present here PhysiPKPD, an open source PhysiCell-based package that allows users to include PKPD in PhysiCell models. Availability & ImplementationThe source code for PhysiPKPD is located here: https://github.com/drbergman/PhysiPKPD.

Decapentaplegic ligand ensures niche space restriction inside and outside of Drosophila testicular niche

Ridwan, S. M. — 2022-09-15

Drosophila male germline stem cells (GSCs) reside at the tip of the testis and surround a cluster of niche cells. It has been believed that the niche-derived Decapentaplegic (Dpp) has a role in maintaining stem cells in close proximity but has no role in the differentiating cells spaced one-cell layer away. However, the range of Dpp diffusion has never been tested. Here, using genetically encoded nanobodies called Morphotrap, we physically block Dpp diffusion without interfering with niche-stem cell signaling and find that diffusible fraction of Dpp is required to ensure differentiation of GSC daughter cells, opposite of its role in maintenance of GSC in the niche. Our work provides an example in which a soluble niche ligand induces opposed cellular responses in stem cells and in differentiating descendants so that the niche can tightly restrict its space. This may be a common mechanism to regulate tissue homeostasis. One sentence summaryBMP ligand diffuses from the niche and has dual, and opposite roles on stem cells and differentiating daughter cells.

Transcription factor TFII-I fine tunes innate properties of B lymphocytes

Singh, A. — 2022-09-22

The ubiquitously expressed transcription factor TFII-I is a multifunctional protein with pleiotropic roles in gene regulation. TFII-I associated polymorphisms are implicated in Sjogrens syndrome and Lupus in humans and, germline deletion of the Gtf2i gene in mice leads to embryonic lethality. Here we report a unique role for TFII-I in homeostasis of innate properties of B lymphocytes. Loss of Gtf2i in murine B lineage cells leads to a change in transcriptome and chromatin landscape, which resembles myeloid-like features and coincides with enhanced sensitivity to LPS induced transcription. TFII-I deficient B cells also show increased switching to IgG3, a phenotype associated with inflammation. These results demonstrate a role for TFII-I in maintaining immune homeostasis and provide clues for GTF2I polymorphisms associated with B cell dominated autoimmune diseases in humans.

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.

DELE1 oligomerization promotes integrated stress response activation

Yang, J. — 2022-10-02

Mitochondria are dynamic organelles that must continually adapt and respond to cellular stress. Recent studies demonstrated that mitochondrial stress can be relayed from mitochondria to the cytosol by the release of a C-terminal proteolytic fragment of DELE1 that binds to the eIF2 kinase HRI to initiate integrate stress response (ISR) signaling. Here, we report the cryo-electron microscopy structure of the active, C-terminal cleavage product of human DELE1 at[~] 3.8 A{degrees} resolution. Our structure reveals that DELE1 assembles into a high-order oligomer that is observed both in vitro and in mammalian cells. Structurally, the oligomer consists of eight DELE1 monomers that assemble with D4 symmetry via two sets of distinct hydrophobic inter-subunit interactions. We identified the key residues involved in DELE1 oligomerization, and confirmed their role in stabilizing the octamer in vitro and in cells using mutagenesis. Further, we show that assembly impaired DELE1 mutants are compromised in their ability to induce ISR activation in cell culture models. Together, our findings provide molecular insights into the activity of DELE1 and how it signals to promote ISR activity following mitochondrial insult.

Differential chondrogenic differentiation between iPSC-derived from healthy and OA cartilage is associated with changes in epigenetic and metabolic transcriptomic signatures

Khan, M. N. — 2022-10-14

Induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine. The iPSCs exhibit a preference for lineage differentiation to the donor cell type indicating the existence of memory of origin. Although the intrinsic effect of the donor cell type on differentiation of iPSCs is well recognized, whether disease-specific factors of donor cells influence the differentiation capacity of iPSC remains unknown. Using viral based reprogramming, we demonstrated the generation of iPSCs from chondrocytes isolated from healthy (AC-iPSCs) and osteoarthritis cartilage (OA-iPSCs). These reprogrammed cells acquired markers of pluripotency and differentiated into uncommitted-mesenchymal progenitors. Interestingly, AC-iPSCs exhibited enhanced chondrogenic potential as compared OA-iPSCs and showed increased expression of chondrogenic genes. Pan-transcriptome analysis showed that chondrocytes derived from AC-iPSCs were enriched in molecular pathways related to energy metabolism and epigenetic regulation, together with distinct expression signature that distinguishes them from OA-iPSCs. The molecular tracing data demonstrated that epigenetic and metabolic marks were imprint of original cell sources from healthy and OA-chondrocytes. Our results suggest that the epigenetic and metabolic memory of disease may predispose OA-iPSCs for their reduced chondrogenic differentiation and thus regulation at epigenetic and metabolic level may be an effective strategy for controlling the chondrogenic potential of iPSCs.

Beyond analytic solution: analysis of FRAP experiments by spatial simulation of the forward problem

Cowan, A. E. — 2023-03-05

Fluorescence redistribution after photobleaching (FRAP) is a commonly used method to understand the dynamic behavior of molecules within cells. Analytic solutions have been developed for specific, well-defined models of dynamic behavior in idealized geometries, but these solutions are inaccurate in complex geometries or when complex binding and diffusion behaviors exist. We demonstrate the use of numerical reaction-diffusion simulation approaches using the easily accessible Virtual Cell (VCell) software, to establish methods for analyzing photobleaching data. We show how multiple simulations employing parameter scans and varying bleaching locations and sizes can help to bracket diffusion coefficients and kinetic rate constants. This approach is applied to problems in membrane surface diffusion, diffusion and binding in cytosolic volumes in complex cell geometries, and analysis of diffusion and binding in intracellular liquid droplets. Statement of SignificanceFluorescence Redistribution After Photobleaching (FRAP) is a widely used experimental method that can reveal important parameters for reaction/diffusion events within cells. However, analytic methods to analyze FRAP experiments are limited to specific geometries and conditions. We demonstrate how spatial numerical simulation methods using the freely available software Virtual Cell can be used to obtain parameter information from FRAP experiments in situations that are not amenable to analytic solutions and that are accessible to most bench biologists.

MolClustPy: A Python Package to Characterize Multivalent Biomolecular Clusters

Chattaraj, A. — 2023-03-15

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSSO_SCPLOWUMMARYC_SCPLOWC_ST_ABSLow-affinity interactions among multivalent biomolecules may lead to the formation of molecular complexes that undergo phase transitions to become extra-large clusters. Characterizing the physical properties of these clusters is important in recent biophysical research. Due to weak interactions such clusters are highly stochastic, demonstrating a wide range of sizes and compositions. We have developed a Python package to perform multiple stochastic simulation runs using NFsim (Network-Free stochastic simulator), characterize and visualize the distribution of cluster sizes, molecular composition, and bonds across molecular clusters and individual molecules of different types. AO_SCPLOWVAILABILITYC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWANDC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWIMPLEMENTATIONC_SCPLOWThe software is implemented in Python. A detailed Jupyter notebook is provided to enable convenient running. Code, user guide and examples are freely available at https://molclustpy.github.io/ CO_SCPLOWONTACTC_SCPLOWachattaraj007@gmail.com, blinov@uchc.edu SO_SCPLOWUPPLEMENTARYC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWINFORMATIONC_SCPLOWAvailable at https://molclustpy.github.io/

Dedifferentiating germ cells regain stem-cell specific polarity checkpoint prior to niche reentry

Bener, M. B. — 2023-04-28

In the Drosophila germline stem cell system, maintenance of the stem cell pool requires "dedifferentiation", in which differentiating cells reattach to the niche and reacquire stem cell properties. However, the mechanism of dedifferentiation remains poorly understood. Here, using long-term live imaging, we show that dedifferentiated cells immediately re-enter mitosis with correct spindle orientation after reattachment to the niche. Analysis of cell cycle markers revealed that these dedifferentiating cells are all in G2 phase. In addition, we found that the observed G2 block during dedifferentiation likely corresponds to a centrosome orientation checkpoint (COC), a previously reported polarity checkpoint. We show that re-activation of a COC is likely required for the dedifferentiation thus ensuring asymmetric division even in dedifferentiated stem cells. Taken together, our study demonstrates the remarkable ability of dedifferentiating cells to reacquire the ability to divide asymmetrically.

Transcranial real-time in vivo recording of electrophysiological neural activity in the rodent brain with near-infrared photoacoustic voltage-sensitive dye imaging

Kang, J. — 2017-10-16

Minimally-invasive monitoring of electrophysiological neural activities in real-time--that enables quantification of neural functions without a need for invasive craniotomy and the longer time constants of fMRI and PET--presents a very challenging yet significant task for neuroimaging. In this paper, we present in vivo functional PA (fPA) imaging of chemoconvulsant rat seizure model with intact scalp using a fluorescence quenching-based cyanine voltage-sensitive dye (VSD) characterized by a lipid vesicle model mimicking different levels of membrane potential variation. The framework also involves use of a near-infrared VSD delivered through the blood-brain barrier (BBB), opened by pharmacological modulation of adenosine receptor signaling. Our normalized time-frequency analysis presented in vivo VSD response in the seizure group significantly distinguishable from those of the control groups at sub-mm spatial resolution. Electroencephalogram (EEG) recording confirmed the changes of severity and frequency of brain activities, induced by chemoconvulsant seizures of the rat brain. The findings demonstrate that the near-infrared fPA VSD imaging is a promising tool for in vivo recording of brain activities through intact scalp, which would pave a way to its future translation.

Evidence against tetrapod-wide digit identities and for a limited frame shift in bird wings

Stewart, T. A. — 2017-12-05

In crown group tetrapods, individual digits are homologized in relation to a pentadactyl ground plan. However, testing hypotheses of digit homology is challenging because it is unclear whether digits develop with distinct and conserved gene regulatory states. Here we show dramatic evolutionary dynamism in the gene expression profiles of digits, challenging the notion that five digit identities are conserved across amniotes. Transcriptomics of developing limbs shows diversity in the patterns of genetic differentiation of digits, although the anterior-most digit of the pentadactyl limb has a unique, conserved expression profile. Further, we identify a core set of transcription factors that are differentially expressed among the digits of amniote limbs; their spatial expression domains, however, vary between species. In light of these results, we reevaluate the frame shift hypothesis of avian wing evolution and conclude that only the identity of the anterior-most digit has shifted position, suggesting a 1,3,4 digit identity in the bird wing.

GABAergic deficits and schizophrenia-like behaviors in a mouse model carrying patient-derived neuroligin-2 R215H mutation

Chen, G. — 2017-11-27

Schizophrenia (SCZ) is a severe mental disorder characterized by delusion, hallucination, and cognitive deficits. We have previously identified from schizophrenia patients a loss-of-function mutation Arg215 [->] His215 (R215H) of neuroligin 2 (NLGN2) gene, which encodes a cell adhesion molecule critical for GABAergic synapse formation and function. Here, we generated a novel transgenic mouse line with neuroligin-2 (NL2) R215H mutation, which showed a significant loss of NL2 protein, reduced GABAergic transmission, and impaired hippocampal activation. Importantly, R215H KI mice displayed anxiety-like behaviors, impaired pre-pulse inhibition (PPI), cognition deficits and abnormal stress responses, recapitulating several key aspects of schizophrenia-like behavior. Our results demonstrate a significant impact of a single point mutation NL2 R215H on brain functions, providing a novel animal model for the study of schizophrenia and neuropsychiatric disorders.

The Innate Immune Response to Ischemic Injury: a Multiscale Modeling Perspective

Dimitrova, E. — 2018-01-17

BackgroundCell death as a result of ischemic injury triggers powerful mechanisms regulated by germline-encoded Pattern Recognition Receptors (PRRs) with shared specificity that recognize invading pathogens and endogenous ligands released from dying cells, and as such are essential to human health. Alternatively, dysregulation of these mechanisms contributes to extreme inflammation, deleterious tissue damage and impaired healing in various diseases. The Toll-like receptors (TLRs) are a prototypical family of PRRs that may be powerful anti-inflammatory targets if agents can be designed that antagonize their harmful effects while preserving host defense functions. This requires an understanding of the complex interactions and consequences of targeting the TLR-mediated pathways as well as technologies to analyze and interpret these, which will then allow the simulation of perturbations targeting specific pathway components, predict potential outcomes and identify safe and effective therapeutic targets.nnResultsWe constructed a multiscale mathematical model that spans the tissue and intracellular scales, and captures the consequences of targeting various regulatory components of injury-induced TLR4 signal transduction on potential pro-inflammatory or pro-healing outcomes. We applied known interactions to simulate how inactivation of specific regulatory nodes affects dynamics in the context of injury and to predict phenotypes of potential therapeutic interventions. We propose rules to link model behavior to qualitative estimates of pro-inflammatory signal activation, macrophage infiltration, production of reactive oxygen species and resolution. We tested the validity of the model by assessing its ability to reproduce published data not used in its construction.nnConclusionsThese studies will enable us to form a conceptual framework focusing on TLR4-mediated ischemic repair to assess potential molecular targets that can be utilized therapeutically to improve efficacy and safety in treating ischemic/inflammatory injury.

mol2sphere: Spherical Decomposition of Multi-Domain Molecules for Visualization and Coarse Grained Spatial Modeling

Masison, J. — 2018-01-31

SummaryProteins, especially those involved in signaling pathways are composed of functional modules consisting of long strings of amino acids. These functional "domains" are linked together in geometric arrangements that can be rigid or flexible, depending on the nature of the linker domains. To understand the structure-function relationships in these macromolecules, it would be helpful to visualize the geometric arrangement of domains. Furthermore, accurate spatial representation of domain structure is necessary for coarse-grain models of the multi-molecular interactions that comprise signaling pathways. Here we introduce a new tool, mol2sphere, that transforms the atomistic structure of a macromolecule into a series of linked spheres corresponding to domains. mol2sphere does this with a k-means clustering algorithm. It may be used for visualization or for coarse grain modeling and simulation.nnAvailability and implementationmol2sphere is available as both a plugin for PyMOL and as a new feature within the SpringSaLaD modeling and simulation software. They run on all desktop platforms and are available with documentation at, respectively, https://nmrbox.org/registry/mol2sphere and http://vcell.org/ssalad. Source code is available for the PyMOL (and python) implementations on the NMRbox platform and for the SpringSaLaD implementation at https://github.com/jmasison/SpringSaLaD.

A normalized drug response metric improves accuracy and consistency of drug sensitivity quantification in cell-based screening

Gupta, A. — 2018-02-08

Accurate quantification of drug effects is crucial for identifying pharmaceutically actionable cancer vulnerabilities. Current cell viability-based measurements often lead to biased response estimates due to varying growth rates and experimental artifacts that explain part of the inconsistency in high-throughput screening results. We developed an improved drug scoring model, normalized drug response (NDR), which makes use of both positive and negative control conditions to account for differences in cell growth rates and experimental noise to better characterize drug-induced effects. We demonstrate an improved consistency and accuracy of NDR compared to existing metrics in assessing drug responses of cancer cells in various culture models and experimental setups. Notably, NDR reliably captures both toxicity and viability responses, and differentiates a wider spectrum of drug behavior, including lethal, growth-inhibitory and growth-stimulatory modes, based on a single viability readout. The method will therefore substantially reduce the time and resources required in cell-based drug sensitivity screening.

COMPUTING SIGNAL TRANSDUCTION IN SIGNALING NETWORKS MODELED AS BOOLEAN NETWORKS, PETRI NETS AND HYPERGRAPHS

Sordo Vieira, — 2018-02-26

Mathematical frameworks circumventing the need of mechanistic detail to build models of signal transduction networks include graphs, hypergraphs, Boolean Networks, and Petri Nets. Predicting how a signal transduces in a signaling network is essential to understand cellular functions and disease. Different formalisms exist to describe how a signal transduces in a given intracellular signaling network represented in the aforementioned modeling frameworks: elementary signaling modes, T-invariants, extreme pathway analysis, elementary flux modes, and simple paths. How do these formalisms compare?nnWe present an overview of how signal transduction networks have been modelled using graphs, hypergraphs, Boolean Networks, and Petri Nets in the literature. We provide a review of the different formalisms for capturing signal transduction in a given model of an intracellular signaling network. We also discuss the existing translations between the different modeling frameworks, and the relationships between their corresponding signal transduction representations that have been described in the literature. Furthermore, as a new formalism of signal transduction, we show how minimal functional routes proposed for signaling networks modeled as Boolean Networks can be captured by computing topological factories, a methodology found in the metabolic networks literature. We further show that in the case of signaling networks represented with an acyclic B-hypergraph structure, the definitions are equivalent. In signaling networks represented as directed graphs, it has been shown that computations of elementary modes via its incidence matrix correspond to computations of simple paths and feedback loops. We show that computing elementary modes based on the incidence matrix of a B-hypergraph fails to capture minimal functional routes.

Comparative Analysis of Linear and Nonlinear Dimension Reduction Techniques on Mass Cytometry Data

Konstorum, A. — 2018-03-01

Mass cytometry, also known as CyTOF, is a newly developed technology for quantification and classification of immune cells that can allow for analysis of over three dozen protein markers per cell. The high dimensional data that is generated requires innovative methods for analysis and visualization. We conducted a comparative analysis of four dimension reduction techniques - principal component analysis (PCA), isometric feature mapping (Isomap), t-distributed stochastic neighbor embedding (t-SNE), and Diffusion Maps by implementing them on benchmark mass cytometry data sets. We compare the results of these reductions using computation time, residual variance, a newly developed comparison metric we term neighborhood proportion error (NPE), and two-dimensional visualizations. We find that t-SNE and Diffusion Maps are the two most effective methods for preserving relationships of interest among cells and providing informative visualizations. In low dimensional embeddings, t-SNE exhibits well-defined phenotypic clustering. Additionally, Diffusion Maps can represent cell differentiation pathways with long projections along each diffusion component. We thus recommend a complementary approach using t-SNE and Diffusion Maps in order to extract diverse and informative cell relationship information in a two-dimensional setting from CyTOF data.

The marked diversity of unique cortical enhancers enables the generation of neuron-specific tools by Enhancer-Driven Gene Expression (EDGE)

Blankvoort, S. — 2018-03-05

Understanding neural circuit function requires individually addressing their component parts: specific neuronal cell types. However, not only do the precise genetic mechanisms specifying neuronal cell types remain obscure, access to these neuronal cell types by transgenic techniques also remains elusive. While most genes are expressed in the brain, the vast majority are expressed in many different kinds of neurons, suggesting that promoters alone are not sufficiently specific to distinguish cell types. However, there are orders of magnitude more distal genetic cis-regulatory elements controlling transcription (i.e. enhancers), so we screened for enhancer activity in microdissected samples of mouse cortical subregions. This identified thousands of novel putative enhancers, many unique to particular cortical subregions. Pronuclear injection of expression constructs containing such region-specific enhancers resulted in transgenic lines driving expression in distinct sets of cells specifically in the targeted cortical subregions, even though the parent genes promoter was relatively nonspecific. These data showcase the promise of utilizing the genetic mechanisms underlying the specification of diverse neuronal cell types for the development of genetic tools potentially capable of targeting any neuronal circuit of interest, an approach we call Enhancer-Driven Gene Expression (EDGE).nnHighlightsO_LIEnhancer ChIP-seq of cortical subregions reveals 59372 putative enhancers.nC_LIO_LI3740 of these are specific to particular cortical subregions.nC_LIO_LIThis reflects the remarkable anatomical diversity of the adult cortex.nC_LIO_LIUnique enhancers provide a means to make targeted cell-type specific genetic tools.nC_LI

Hyperexcitable phenotypes in iPSC-derived neurons from patients with 15q11-q13 duplication syndrome, a genetic form of autism

Fink, J. J. — 2018-03-21

Chromosome 15q11-q13 duplication syndrome (Dup15q) is a neurogenetic disorder caused by duplications of the maternal copy of this region. In addition to hypotonia, motor deficits, and language impairments, Dup15q patients commonly meet the criteria for autism spectrum disorder (ASD) and have a high prevalence of seizures. Here, we explored mechanisms of hyperexcitability in neurons derived from induced pluripotent stem cell (iPSC) lines from Dup15q patients. Maturation of resting membrane potential in Dup15q-derived neurons was similar to neurons from unaffected control subjects, but Dup15q neurons had delayed action potential maturation and increased synaptic event frequency and amplitude. Dup15q neurons also showed impairments in activity-dependent synaptic plasticity and homeostatic synaptic scaling. Finally, Dup15q neurons showed an increased frequency of spontaneous action potential firing compared to control neurons, in part due to disruption of KCNQ2 channels. Together these data point to multiple mechanisms underlying hyperexcitability that may provide new targets for the treatment of seizures and other phenotypes associated with Dup15q.

An important role for periplasmic storage in Pseudomonas aeruginosa copper homeostasis revealed by a combined experimental and computational modeling study

Parmar, J. H. — 2018-04-13

Biological systems require precise copper homeostasis enabling metallation of cuproproteins while preventing metal toxicity. In bacteria, sensing, transport and storage molecules act in coordination to fulfill these roles. However, there is not yet a kinetic schema explaining the system integration. Here, we report a model emerging from experimental and computational approaches that describes the dynamics of copper distribution in Pseudomonas aeruginosa. Based on copper uptake experiments, a minimal kinetic model describes well the copper distribution in the wild type bacteria but is unable to explain the behavior of the mutant strain lacking CopA1, a key Cu+ efflux ATPase. The model was expanded through an iterative hypothesis-driven approach, arriving to a mechanism that considers the induction of compartmental pools and the parallel function of CopA and Cus efflux systems. Model simulations support the presence of a periplasmic copper storage with a crucial role under dyshomeostasis conditions in P. aeruginosa. Importantly, the model predicts not only the interplay of periplasmic and cytoplasmic pools but also the existence of a threshold in the concentration of external copper beyond which cells lose their ability to control copper levels.

Transcranial photoacoustic imaging of NMDA-evoked focal circuit dynamics in rat forebrain

Kang, J. — 2018-04-26

Transcranial functional photoacoustic (fPA) voltage-sensitive dye (VSD) imaging promises to overcome current temporal and spatial limitations of current neuroimaging modalities. The technique previously distinguished global seizure activity from control neural activity in groups of rats. To validate the focal specificity of transcranial fPA neuroimaging in vivo, we now present proofs-of-concept that the results differentiate between low- and high-dose N-methyl-D-aspartate (NMDA) evoked neural activity in rat hippocampus. Concurrent quantitative EEG (qEEG) and microdialysis recorded real-time circuit dynamics and glutamate concentration change, respectively. We hypothesized that location-specific fPA VSD contrast would identify the neural dynamics in hippocampus with the correlation to NMDA evoked focal glutamate release and time-specific EEG signals. To test the hypothesis, we infused 0.3 to 3.0 mM NMDA at 2 l/min over 60 min via an implanted microdialysis probe. The dialysate samples collected every 20 min during the infusion were analyzed for focal changes in extracellular glutamate release. Transcranial fPA VSD imaging provided NMDA-evoked VSD responses with positive correlation to extracellular glutamate concentration change at the contralateral side of the microdialysis probe. The graded response represents the all-or-none gating system of the dentate gyrus (DG) in hippocampus. Quantitative EEG (qEEG) successfully confirmed induction of focal seizure activity during NMDA infusion. We conclude that transcranial fPA VSD imaging distinguished graded DG gatekeeping functions, based on the VSD redistribution mechanism sensitive to electrophysiologic membrane potential. The results suggest the potential future use of this emerging technology in clinics and science as an innovative and significant functional neuroimaging modality.

Sequence variation of rare outer membrane protein β-barrel domains in clinical strains provides insights into the evolution of Treponema pallidum subsp. pallidum, the syphilis spirochete.

Kumar, S. — 2018-05-09

In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum (TPA), the syphilis spirochete, and identifying its surface-exposed {beta}-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of {beta}-barrel-encoding regions of tprC, tprD, and bamA, in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used tp0548 genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) {beta}-barrels in clinical strains could be grouped according to allelic variants in TPA reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored {beta}-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. Based upon structural models, non-conservative amino acid substitutions in predicted transmembrane {beta}-strands of TprC and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive TPA OMP diversity and that genetic exchange contributes to the evolutionary biology of TPA. They also set the stage for topology-based analysis of antibody responses against OMPs and help frame strategies for syphilis vaccine development.nnIMPORTANCEDespite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum (TPA), little is known about how their surface-exposed, {beta}-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the {beta}-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in TPA from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within {beta}-barrel domains occurred predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function also were noted. Our findings suggest that selection pressures exerted by human populations drive TPA OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of TPA and frame strategies for vaccine development based upon conserved OMP extracellular loops.

A computational model to understand mouse iron physiology and diseases

Parmar, J. H. — 2018-05-16

It is well known that iron is an essential element for life but is toxic when in excess or in certain forms. Accordingly there are many diseases that result directly from either lack or excess of iron. Iron has also been associated with a wide range of other diseases and may have an important role in aging. Yet many molecular and physiological aspects of iron regulation have only been discovered recently and others are still awaiting elucidation. In the last 18 years, after the discovery of the hormone hepcidin, many details of iron regulation have become better understood and a clearer picture is starting to emerge, at least in qualitative terms. However there is still no good quantitative and dynamic description of iron absorption, distribution, storage and mobilization that agrees with the wide array of phenotypes presented in several iron-related diseases. The present work addresses this issue by developing a mathematical model of iron distribution in mice that was calibrated with existing ferrokinetic data and subsequently validated against data from a series of iron disorders, such as hemochromatosis, {beta}-thalassemia, atransferrinemia and anemia of inflammation. To adequately fit the ferrokinetic data required including the following mechanisms: a) the role of transferrin in deliving iron to tissues, b) the induction of hepcidin by high levels of transferrin-bound iron, c) the ferroportin-dependent hepcidin-regulated iron export from tissues, d) the erythropoietin regulation of erythropoiesis, and e) direct NTBI uptake by the liver. The utility of such a model to simulate disease interventions was demonstrated by using it to investigate the outcome of different schedules of transferrin treatment in {beta}-thalassemia. The present model is a successful step towards a comprehensive mathematical model of iron physiology incorporating cellular and organ level details.

Examining the Role of the Surfactant Family Member SFTA3 in Interneuron Specification

Chen, C. — 2018-05-24

The transcription factor NKX2.1, expressed at high levels in the medial ganglionic eminence (MGE), is a master regulator of cortical interneuron progenitor development. To identify gene candidates with expression profiles similar to NKX2.1, previous transcriptome analysis of human embryonic stem cell (hESC)-derived MGE-like progenitors revealed SFTA3 as the strongest candidate. Quantitative real-time PCR analysis of hESC-derived NKX2.1-positive progenitors and transcriptome data available from the Allen Institute for Brain Science revealed comparable expression patterns for NKX2.1 and SFTA3 during interneuron differentiation in vitro and demonstrated high SFTA3 expression in the human MGE. Although SFTA3 has been well studied in the lung, the possible role of this surfactant protein in the MGE during embryonic development remains unexamined. To determine if SFTA3 plays a role in MGE specification, SFTA3-/- and NKX2.1 -/-hESC lines were generated using custom designed CRISPRs. We show that NKX2.1 KOs have a significantly diminished capacity to differentiate into MGE interneuron subtypes. SFTA3 KOs also demonstrated a somewhat reduced ability to differentiate down the MGE-like lineage, although not as severe relative to NKX2.1 deficiency. These results suggest NKX2.1 and SFTA3 are co-regulated genes, and that deletion of SFTA3 does not lead to a major change in the specification of MGE derivatives.

Three-dimensional organization of transzonal projections and other cytoplasmic extensions in mouse ovarian follicles

Baena, V. — 2018-06-20

Each mammalian oocyte is nurtured by its own multi-cellular structure, the ovarian follicle. We used new methods for serial section electron microscopy to examine entire cells and their projections in mouse antral ovarian follicles. It is already known that cumulus cells send towards the oocyte thin cytoplasmic projections called transzonal projections (TZPs), which are crucial for normal oocyte development. We found that most TZPs do not reach the oocyte, and that they often branch and make gap junctions with each other. Furthermore, the connected TZPs are usually contacted on their shaft by oocyte microvilli. Mural granulosa cells were found to possess randomly oriented cytoplasmic projections that are strikingly similar to free-ended TZPs. We propose that granulosa cells use cytoplasmic projections to search for the oocyte, and cumulus cell differentiation results from a contact-mediated paracrine interaction with the oocyte.

Incomplete penetrance for isolated congenital asplenia in humans with mutations in translated and untranslated RPSA exons

Bolze, A. — 2018-06-27

Isolated congenital asplenia (ICA) is the only known human developmental defect exclusively affecting a lymphoid organ. In 2013, we showed that private deleterious mutations in the protein-coding region of RPSA, encoding ribosomal protein SA, caused ICA by haploinsufficiency with complete penetrance. We reported seven heterozygous protein-coding mutations in 8 of the 23 kindreds studied, including 6 of the 8 multiplex kindreds. We have since enrolled 33 new kindreds, 5 of which are multiplex. We describe here eleven new heterozygous ICA-causing RPSA protein-coding mutations, and the first two mutations in the 5-UTR of this gene, which disrupt mRNA splicing. Overall, 40 of the 73 ICA patients (55%) and 23 of the 56 kindreds (41%) carry mutations located in translated or untranslated exons of RPSA. Eleven of the 43 kindreds affected by sporadic disease (26%) carry RPSA mutations, whereas 12 of the 13 multiplex kindreds (92%) carry RPSA mutations. We also report that six of eighteen (33%) protein-coding mutations and the two (100%) 5-UTR mutations display incomplete penetrance. Three mutations were identified in 2 independent kindreds, due to a hotspot or a founder effect. Lastly, RPSA ICA-causing mutations were demonstrated to be de novo in 7 of the 23 probands. Mutations in RPSA exons can affect the translated or untranslated regions and can underlie ICA with complete or incomplete penetrance.

A biophysical model of molecular clusters: interplay of multivalency, membrane localization, steric hindrance, molecular flexibility and intracellular crowding

Chattaraj, A. — 2018-07-20

Dynamic molecular clusters are assembled through weak multivalent interactions and are platforms for cellular functions, especially receptor-mediated signaling. Clustering is also a prerequisite for liquid-liquid phase separation. But it is not well understood how molecular structure and cellular organization control clustering. Using coarse-grain kinetic Langevin dynamics, we performed computational experiments on a prototypical ternary system modeled after membrane-bound nephrin, the adaptor Nck1 and the actin nucleation promoting factor NWASP. Steady state cluster size distributions favored stoichiometries that optimized binding (stoichiometry matching), but still were quite broad. At high concentrations, the system can be driven beyond the saturation boundary such that cluster size is limited only by the number of available molecules. This behavior would be predictive of phase separation. Domains close to binding sites sterically inhibited clustering much less than terminal domains because the latter effectively restrict access to the cluster interior. Increased flexibility of interacting molecules diminished clustering by shielding binding sites within compact conformations. Membrane association of nephrin increased the cluster size distribution in a density-dependent manner. These properties provide insights into how molecular ensembles function to localize and amplify cell signaling.

Benchmarking Time-Series Data Discretization on Inference Methods

Li, Y. — 2018-08-01

The rapid development in quantitatively measuring DNA, RNA, and protein has generated a great interest in the development of reverse-engineering methods, that is, data-driven approaches to infer the network structure or dynamical model of the system. Many reverse-engineering methods require discrete quantitative data as input, while many experimental data are continuous. Some studies have started to reveal the impact that the choice of data discretization has on the performance of reverse-engineering methods. However, more comprehensive studies are still greatly needed to systematically and quantitatively understand the impact that discretization methods have on inference methods. Furthermore, there is an urgent need for systematic comparative methods that can help select between discretization methods. In this work, we consider 4 published intracellular networks inferred with their respective time-series datasets. We discretized the data using different discretization methods. Across all datasets, changing the data discretization to a more appropriate one improved the reverse-engineering methods performance. We observed no universal best discretization method across different time-series datasets. Thus, we propose DiscreeTest, a two-step evaluation metric for ranking discretization methods for time-series data. The underlying assumption of DiscreeTest is that an optimal discretization method should preserve the dynamic patterns observed in the original data across all variables. We used the same datasets and networks to show that DiscreeTest is able to identify an appropriate discretization among several candidate methods. To our knowledge, this is the first time that a method for benchmarking and selecting an appropriate discretization method for time-series data has been proposed.nnAvailabilityAll the datasets, reverse-engineering methods and source code used in this paper are available in Vera-Liconas lab Github repository: https://github.com/VeraLiconaResearchGroup/Benchmarking_TSDiscretizations

Alcohol-responsive genes identified in human iPSC-derived neural cultures

Jensen, K. — 2018-07-31

Alcohol use contributes to numerous diseases and injuries. The nervous system is affected by alcohol in diverse ways, though the molecular mechanisms of these effects are not clearly understood. Using human-induced pluripotent stem cells (iPSCs), we developed a neural cell culture model to identify the mechanisms of alcohols effects. iPSCs were generated from fibroblasts and differentiated into forebrain neural cells cultures that were treated with 50 mM alcohol or sham conditions (same media lacking alcohol) for 7 days. We analyzed gene expression using total RNA sequencing (RNA-seq) for 34 samples derived from 11 subjects and for 10 samples from 5 subjects in an independent experiment that had intermittent exposure to the same dose of alcohol. We also analyzed genetic effects on gene expression and conducted a weighted correlation network analysis. We found that differentiated neural cell cultures have the capacity to recapitulate gene regulatory effects previously observed in specific primary neural tissues and identified 226 genes that were differentially expressed (FDR< 0.1) after alcohol treatment. The effects on expression included decreases in INSIG1 and LDLR, two genes involved in cholesterol homeostasis. We also identified a module of 58 co-expressed genes that were uniformly decreased following alcohol exposure. The majority of these effects were supported in the independent alcohol exposure experiment. Enrichment analysis linked the alcohol responsive genes to cell cycle, notch signaling, and cholesterol biosynthesis pathways, which are disrupted in several neurological disorders. Our findings suggest that there is convergence between these disorders and the effects of alcohol exposure.

Neuregulin 1 type III reduces severity in a mouse model of Congenital Hypomyelinating Neuropathy

Sophie, B. — 2018-08-07

Myelin sheath thickness is precisely regulated and essential for rapid propagation of action potentials along myelinated axons. In the peripheral nervous system, extrinsic signals from the axonal protein neuregulin 1 type III regulate Schwann cell fate and myelination. Here we ask if modulating neuregulin 1 type III levels in neurons would restore myelination in a model of congenital hypomyelinating neuropathy (CHN). Using a mouse model of CHN, we rescued the myelination defects by early overexpression of neuregulin 1 type III. Surprisingly, the rescue was independent from the upregulation of Egr2 or essential myelin genes. Rather, we observed the activation of MAPK/ERK and other myelin genes such as peripheral myelin protein 2 (Pmp2) and oligodendrocyte myelin glycoprotein (Omg). We also confirmed that the permanent activation of MAPK/ERK in Schwann cells has detrimental effects on myelination. Our findings demonstrate that the modulation of axon-to-glial neuregulin 1 type III signaling has beneficial effects and restores myelination defects during development in a model of CHN.nnnnO_TBL View this table:norg.highwire.dtl.DTLVardef@103281aorg.highwire.dtl.DTLVardef@3dc68eorg.highwire.dtl.DTLVardef@1b6c5feorg.highwire.dtl.DTLVardef@80e419org.highwire.dtl.DTLVardef@1ff2591_HPS_FORMAT_FIGEXP M_TBL C_TBL

A bipartite boundary element restricts UBE3A imprinting to mature neurons.

Hsiao, J. S. — 2018-08-31

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of function from the maternal allele of UBE3A, a gene encoding an E3 ubiquitin ligase. UBE3A is only expressed from the maternally-inherited allele in mature human neurons due to tissue-specific genomic imprinting. Imprinted expression of UBE3A is restricted to neurons by expression of UBE3A antisense transcript (UBE3A-ATS) from the paternally-inherited allele, which silences the paternal allele of UBE3A in cis. However, the mechanism restricting UBE3A-ATS expression and UBE3A imprinting to neurons is not understood. We used CRISPR/Cas9-mediated genome editing to functionally define a bipartite boundary element critical for neuron-specific expression of UBE3A-ATS in humans. Removal of this element led to upregulation of UBE3A-ATS without repressing paternal UBE3A. However, increasing expression of UBE3A-ATS in the absence of the boundary element resulted in full repression of paternal UBE3A, demonstrating that UBE3A imprinting requires both the loss of function from the boundary element as well as upregulation of UBE3A-ATS. These results suggest that manipulation of the competition between UBE3A-ATS and UBE3A may provide a potential therapeutic approach for AS.nnSIGNIFICANCE STATEMENTAngelman syndrome is a neurodevelopmental disorder caused by loss of function from the maternal allele of UBE3A, an imprinted gene. The paternal allele of UBE3A is silenced by a long, non-coding antisense transcript in mature neurons. We have identified a boundary element that stops the transcription of the antisense transcript in human pluripotent stem cells, and thus restricts UBE3A imprinted expression to neurons. We further determined that UBE3A imprinting requires both the loss of the boundary function and sufficient expression of the antisense transcript to silence paternal UBE3A. These findings provide essential details about the mechanisms of UBE3A imprinting that may suggest additional therapeutic approaches for Angelman syndrome.

A contraction stress model of hypertrophic cardiomyopathy due to thick filament sarcomere mutations

Cohn, R. — 2018-09-04

Thick filament sarcomere mutations are the most common cause of hypertrophic cardiomyopathy (HCM), a disorder of heart muscle thickening associated with sudden cardiac death and heart failure, with unclear mechanisms. We engineered an isogenic panel of four human HCM induced pluripotent stem cell (iPSc) models using CRISPR/Cas9, and studied iPSc-derived cardiomyocytes (iCMs) in 3-dimensional cardiac microtissue (CMT) assays that resemble in vivo cardiac architecture and biomechanics. HCM mutations result in hypercontractility in association with prolonged relaxation kinetics in proportion to mutation pathogenicity but not calcium dysregulation. RNA sequencing and protein expression studies identified that HCM mutations result in p53 activation secondary to increased oxidative stress, which results in increased cytotoxicity that can be reversed by p53 genetic ablation. Our findings implicate hypercontractility as an early consequence of thick filament mutations, and the p53 pathway as a molecular marker and candidate therapeutic target for thick filament HCM.

DksA controls the response of the Lyme disease spirochete Borrelia burgdorferi to starvation

Boyle, W. K. — 2018-09-20

The pathogenic spirochete Borrelia burgdorferi senses and responds to diverse environmental challenges, including changes in nutrient availability, throughout its enzootic cycle in Ixodes spp. ticks and vertebrate hosts. This study examined the role of DnaK suppressor protein (DksA) in the transcriptional response of B. burgdorferi to starvation. Wild-type and dksA mutant B. burgdorferi strains were subjected to starvation by shifting mid-logarithmic phase cultures grown in BSK II medium to serum-free RPMI medium for 6 h under microaerobic conditions (5% CO2, 3% O2). Microarray analyses of wild-type B. burgdorferi revealed that genes encoding flagellar components, ribosomal proteins, and DNA replication machinery were downregulated in response to starvation. DksA mediated transcriptomic responses to starvation in B. burgdorferi as the dksA-deficient strain differentially expressed only 47 genes in response to starvation compared to the 500 genes differentially expressed in wild-type strains. Consistent with a role for DksA in the starvation response of B. burgdorferi, fewer CFUs were observed for dksA mutant after prolonged starvation in RPMI medium compared to wild-type B. burgdorferi. Transcriptomic analyses revealed a partial overlap between the DksA regulon and the regulon of RelBbu, the guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthetase that controls the stringent response; the DksA regulon also included many plasmid-borne genes. Additionally, the dksA mutant strain exhibited constitutively elevated (p)ppGpp levels compared to the wild-type strain, implying a regulatory relationship between DksA and (p)ppGpp. Together, these data indicate that DksA along with (p)ppGpp direct the stringent response to effect B. burgdorferi adaptation to its environment.nnIMPORTANCEThe Lyme disease bacterium Borrelia burgdorferi must sense and respond to diverse environments as it cycles between its tick vectors and various vertebrate hosts. B. burgdorferi must withstand prolonged periods of starvation while it resides in unfed Ixodes ticks. In this study, the regulatory protein DksA is shown to play a pivotal role controlling the transcriptional responses of B. burgdorferi to starvation. The results of this study suggest that DksA gene regulatory activity impacts B. burgdorferi metabolism, virulence gene expression, and the ability of this bacterium to complete its natural life cycle.

Specification of diverse cell types during early neurogenesis of the mouse cerebellum

Wizeman, J. W. — 2018-10-11

We applied single-cell RNA sequencing to profile genome-wide gene expression in about 9,400 individual cerebellar cells from the mouse embryo at embryonic day 13.5. Reiterative clustering identified the major cerebellar cell types and subpopulations of different lineages. Through pseudotemporal ordering to reconstruct developmental trajectories, we identified novel transcriptional programs controlling cell fate specification of populations arising from the ventricular zone and the anterior rhombic lip, two distinct germinal zones of the embryonic cerebellum. Together, our data revealed cell-specific markers for studying the cerebellum, important specification decisions, and a number of previously unknown subpopulations that may play an integral role in the formation and function of the cerebellum. Importantly, we identified a potential mechanism of vermis formation, which is affected by multiple congenital cerebellar defects. Our findings will facilitate new discovery by providing insights into the molecular and cell type diversity in the developing cerebellum.

The landscape of intrinsic and evolved fluoroquinolone resistance in Acinetobacter baumannii includes suppression of drug-induced prophage replication

Geisinger, E. — 2018-10-14

The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii, the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (Topo IV). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug sensitive and resistant strains to identify loci that alter the sensitivity of the organism to ciprofloxacin. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectrum of mutations that altered drug sensitivity was found to be similar in the drug sensitive and double mutant gyrAparC background having resistance alleles in both genes. In contrast, introduction of a single gyrA resistance allele, resulting in preferential poisoning of Topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential Topo IV poisoning was induction of DNA synthesis in the region of two endogenous prophages, which appeared to occur in situ. Induction of the selective DNA synthesis in the gyrA background was also linked to enhanced activation of SOS response and heightened transcription of prophage genes relative to that observed in either the WT or gyrAparC double mutants. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to suppression of hyperactivation of the SOS response and endogenous prophage DNA synthesis.nnImportanceFluoroquinolones have been extremely successful antibiotics. Their clinical efficacy derives from the ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and Topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we sought to understand how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii, which causes resistant, life-threating infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to lower fluoroquinolone resistance independently of target alteration. Remarkably, fluoroquinolone targeting of Topo IV in specific mutants caused dramatic prophage hyperinduction, a response that was muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities.

Functional Interrogation of Lynch Syndrome Associated MSH2 Missense Variants Using CRISPR-Cas9 Gene Editing in Human Embryonic Stem Cells

Rath, A. — 2018-11-01

Lynch syndrome (LS) is a hereditary cancer predisposition condition caused by inactivating germline mutations in one of the DNA mismatch repair (MMR) genes. Identifying a deleterious germline mutation by DNA sequencing is important for confirming an LS diagnosis. Frameshift and nonsense mutations significantly alter the protein product and likely impair MMR function. However, the implication of a missense mutation is often difficult to interpret. Referred to as variants of uncertain significance (VUS), their discovery hampers the definitive LS diagnosis. To determine the pathogenic significance of a VUS it is helpful to know its impact on protein function. Functional studies in the test tube and in cellular models have been performed for some VUS, however, these studies have been limited by the artificial nature of the assays. We report here an improved functional assay in which we engineered site-specific MSH2 VUS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild-type gene. We then characterized the impact of the variants on cellular MMR functions including DNA damage response signaling upon challenge with a DNA alkylating agent and the repair of DNA microsatellites. We classified the MMR functional capability of 8 of 10 VUS under study providing valuable information for determining their likelihood of being bona fide LS mutations. This improved human cell-based assay system for functionally testing MMR gene VUS will facilitate the identification of high risk LS patients.nnSignificance StatementUnderstanding how cancer-associated missense variants in MMR genes affect function helps determine whether they truly contribute to disease. Laboratory assays previously utilized are limited by their artificial nature. To improve this, we introduced variants directly into the endogenous MMR loci in hESCs using CRISPR-Cas9 gene editing. This approach allows us to assess each variant while being expressed by its normal regulatory elements in a cellular environment. Our results will help guide the management of patients world-wide who carry these variants. At the same time, this study provides a technical road map for assessing the functional effects of all LS-associated variants, as well as variants linked to other genetic diseases where a cell-based functional assay is available.

Identification of proteins involved in Trypanosoma brucei DNA replication fork dynamics using nascent DNA proteomics

Rocha-Granados, M. — 2018-11-12

DNA replication, transcription and chromatin remodeling are coordinated to ensure accurate duplication of genetic and epigenetic information. In regard to DNA replication, trypanosomatid parasites such as Trypanosoma brucei display unusual properties including significantly fewer origins of replication than model eukaryotes, a highly divergent Origin Replication Complex (ORC), and an apparent lack of several replication factor homologs. Although recent studies in T. brucei indicate functional links among DNA replication, transcription, and antigenic variation, the underlying mechanisms remain unknown. Here, we adapted an unbiased technology for the identification of replication fork proteins called iPOND (isolation of proteins on nascent DNA) to T. brucei, its first application to a parasite system. This led to the mass spectrometric identification of core replication machinery and of proteins associated with transcription, chromatin organization, and DNA repair that were enriched in the vicinity of an unperturbed active replication fork. Of a total of 410 enriched proteins, among which DNA polymerase and replication factor C were scoring in the top, around 25% of the proteins identified were of unknown function and, therefore, have the potential to be essential trypanosome-specific replication proteins. Initial characterization of a protein annotated as a Replication Factor C subunit (Tb927.10.7990), and a protein of unknown function (Tb927.3.5370) revealed that both proteins retain nuclear localization throughout the cell cycle. While Tb927.3.5370 appeared to be a dispensable gene, Tb927.10.7990 proved to be essential since its silencing caused a growth defect in procyclic cells, accumulation of zoids and impaired DNA replication. Future studies on the generated proteins list can contribute to the understanding of DNA replication dynamics in T. brucei and how replication is coordinated with other cellular processes to maintain genome integrity.

Tethered Bichromophoric Fluorophore Quencher Voltage Sensitive Dyes

Yan, P. — 2018-11-19

Voltage sensitive dyes (VSDs) are used for in vitro drug screening and for in vivo imaging of patterns of electrical activity. However, wide application of this technology is limited by poor sensitivity. A promising approach uses a 2-component system consisting of charged membrane permeable quenchers together with fluorophores labeling one side of the membrane; this produces voltage-dependent fluorescence quenching. However, to achieve good sensitivity, the quencher compound must be used at high concentrations, which can perturb the membrane capacitance or have other pharmacological effects. By developing tethered bichromophoric fluorophore quencher (TBFQ) dyes, where the fluorophore and quencher are covalently connected by a long hydrophobic chain, the concentration required is minimized, and the sensitivity is maximized. A series of 13 TBFQ dyes based on the AminoNaphthylEthenylPyridinium (ANEP) fluorophore and the dipicrylamine anion (DPA) quencher have been synthesized and tested in an artificial lipid bilayer apparatus. The best one from the screening, TBFQ1, shows a 2.5 fold change in fluorescence per 100mV change in membrane potential, and the response kinetics is in 10-20 ms range. This sensitivity is an order of magnitude better than commonly used fluorescent voltage sensors. The design principles for TBFQ VSDs described here can be readily extended to other spectral regions and promise to greatly enhance our ability to monitor electrical activity in cells and tissues.

Defining developmental diversification of diencephalon neurons through single-cell gene expression profiling

Guo, Q. — 2018-11-28

The embryonic diencephalon gives rise to diverse neuronal cell types, which form complex integration centers and intricate relay stations of the vertebrate forebrain. Prior anecdotal gene expression studies suggest several developmental compartments within the developing diencephalon. In the current study, we utilized single-cell RNA sequencing to profile transcriptomes of dissociated cells from the diencephalon of E12.5 mouse embryos. Through analysis of unbiased transcriptional data, we identified the divergence of different progenitors, intermediate progenitors, and emerging neuronal cell types. After mapping the identified cell groups to their spatial origins, we were able to characterize the molecular features across different cell types and cell states, arising from various diencephalic compartments. Furthermore, we reconstructed the developmental trajectory of different cell lineages within the diencephalon. This allowed the identification of the genetic cascades and gene regulatory networks underlying the progression of the cell cycle, neurogenesis, and cellular diversification. The analysis provides new insights into the molecular mechanism underlying the specification and amplification of thalamic progenitor cells. In addition, the single-cell-resolved trajectories not only confirm a close relationship between the rostral thalamus and prethalamus, but also uncover an unexpected close relationship between the caudal thalamus, epithalamus and rostral pretectum. Our data provide a useful resource for the systematic study of cell heterogeneity and differentiation kinetics within the developing diencephalon.

Molecular Process Diagram: a precise, scalable and compact visualization of rule-based models

Vasilescu, D. — 2018-12-21

Rule-based modeling allows representation and simulation of biological systems where molecular features (such as protein domains) and feature-specific details of molecular interactions are accounted for. The rule-based description is precise and can define very fine molecular details (e.g. how phosphorylation status of a single residue in a multi-protein complex can affect affinity of another binding site of another protein within the same complex), but makes it difficult to effectively combine the assumptions scribed within the multiple rules of a model into a diagrammatic view. Various visualization schemas have been suggested, but they are all highly rule-based centric (e.g. a visual list of unconnected rules, extended contact maps, or atom-rule graphs). None of them match the clarity of traditional reaction/pathway diagrams, where a researcher can easily visually track the transitions and modifications of chemical species occurring in the biological systems being modeled. Here we present a novel approach and software for precise, scalable and compact representation of rule-based models that we call Molecular Process Diagram. It is based on the three basic elements: interacting molecular complexes, molecular sites directly modified by a rule, and molecular sites that are not modified but contribute to a rule mechanism (e.g. a site that in a phosphorylated state changes binding affinity of another site). Multiple levels of resolution are available: pathway-like diagram of interactions among molecules, optional site-specific interactions, and additional contingencies for interactions. Inclusion of molecular sites enables unambiguous reconstruction of the rule descriptions from the visual diagram without additional supporting documentation, while still keeping a pathway-like visual appearance. The proposed approach for visualization has been implemented in the Virtual Cell (VCell) modeling and simulation framework. Our Molecular Process Diagrams extend the notion of Systems Biology Graphical Notation (SBGN) process diagrams and use SBGN-compliant conventions.nnSummaryKinetic models have provided significant insights into biological regulatory mechanisms even though they typically did not take into consideration the details of protein subcomponents such as binding domains and phosphorylation sites. However, these details are often required for an accurate understanding of the events that occur during cell signaling. Without such detailed understanding, intervention strategies to act on signaling pathways in pathological conditions are bound to have limited success. This need to include site-specific details into models led to the advance of rule-based modeling. While rules describe the details of interactions with unmatched precision, they often obscure the "big picture", i.e. a pathway-like description of the information flow through the biological system. An intuitive visual diagram is crucial for understanding the assumptions embodied into a model. Here we present a novel approach and software for precise, scalable and compact representation of rule-based models that we call Molecular Process Diagram. It allows visualizing in a pathway-like diagram of the interacting molecules, the molecular sites modified, and the molecular sites that affect the interactions. The approach is implemented in the Virtual Cell (VCell) modeling and simulation framework and suggested as an extension for the Systems Biology Graphical Notations (SBGN) standard.

Abl signaling shapes the intrinsic fluctuations of actin to direct growth of a pioneer axon in Drosophila

Clarke, A. — 2019-01-04

The fundamental problem in axon growth and guidance is to understand how cytoplasmic signaling modulates the cytoskeleton to produce directed growth cone motility. We show here that the TSM1 pioneer axon of Drosophila extends by using Abl tyrosine kinase to shape the intrinsic fluctuations of a mass of accumulated actin in the distal axon. The actin mass fluctuates stochastically in length, but with a small, forward bias that drives the axon along its trajectory by promoting emergence of protrusions in leading intervals where actin accumulates, and collapse of protrusions in lagging intervals that actin has vacated. The actin mass is sculpted by Abl signaling, which probabilistically modulates its key parameters - its width and internal disorder - to drive its advance, while maintaining internal coherence. Comparison of TSM1 to other systems suggests that the mechanism we demonstrate here is apt to be common among pioneer axons in many organisms.

Simulation of the mechanics of actin assembly during endocytosis in yeast

Nickaeen, M. — 2019-01-11

We formulated a spatially resolved model to estimate forces exerted by a polymerizing actin meshwork on an invagination of the plasma membrane during endocytosis in yeast cells. The model is a continuous approximation tightly constrained by experimental data. Simulations of the model produce forces that can overcome resistance of turgor pressure in yeast cells. Strong forces emerge due to the high density of polymerized actin in the vicinity of the invagination and because of entanglement of the meshwork due to its dendritic structure and crosslinking. The model predicts forces orthogonal to the invagination that would result in a flask shape that diminishes the net force due to turgor pressure. Simulations of the model with either two rings of nucleation promoting factors as in fission yeast or a single ring of nucleation promoting factors as in budding yeast produce enough force to elongate the invagination against the turgor pressure.

Follicle-stimulating hormone and luteinizing hormone increase Ca2+ in the granulosa cells of mouse ovarian follicles

Egbert, J. R. — 2019-01-14

Summary sentenceBoth FSH and LH increase Ca2+ in the granulosa cells of intact ovarian follicles from mice expressing genetically encoded sensors.nnABSTRACTIn mammalian ovarian follicles, follicle stimulating hormone (FSH) and luteinizing hormone (LH) signal primarily through the G-protein Gs to elevate cAMP, but both of these hormones can also elevate Ca2+ under some conditions. Here we investigate FSH- and LH-induced Ca2+ signaling in intact follicles of mice expressing genetically encoded Ca2+ sensors, Twitch-2B and GCaMP6s. At a physiological concentration (1 nM), FSH elevates Ca2+ within the granulosa cells of preantral and antral follicles. The Ca2+ rise begins several minutes after FSH application, peaks at ~10 minutes, remains above baseline for another ~10 minutes, and depends on extracellular Ca2+. However, suppression of the FSH-induced Ca2+ increase by reducing extracellular Ca2+ does not inhibit FSH-induced phosphorylation of MAP kinase, estradiol production, or the acquisition of LH responsiveness. Like FSH, LH also increases Ca2+, when applied to preovulatory follicles. At a physiological concentration (10 nM), LH elicits Ca2+ oscillations in a subset of cells in the outer mural granulosa layer. These oscillations continue for at least 6 hours and depend on the activity of Gq family G-proteins. Suppression of the oscillations by Gq inhibition does not inhibit meiotic resumption, but does slightly attenuate ovulation. In summary, both FSH and LH increase Ca2+ in the granulosa cells of intact follicles, but the functions of these Ca2+ rises are only starting to be identified.

The Evolution of Placental Invasion and Cancer Metastasis are Causally Linked

Gupta, K. — 2019-01-23

Among mammals, the extent of placental invasion is correlated with vulnerability to malignancy. Animals with more invasive placentation (e.g. humans) are more vulnerable to malignancy, whereas animals with a non-invasive placenta (e.g. ruminants) are less likely to develop malignant cancer. To explain this correlation, we propose the hypothesis of Evolved Levels of Invasibility (ELI) positing that the permissiveness of stromal tissue to invasion is a unitary character affecting both placental and cancer invasion. We provide evidence for this hypothesis by contrasting invasion of human and bovine cancer and placental cells into a lawn of stromal cells from different species. We find that both bovine endometrial and skin fibroblasts are more resistant to invasion of placental and cancer cells than their human counterparts. Gene expression profiling identified genes with high expression in human but not bovine fibroblasts. Knocking down of a subset of them in human fibroblasts leads to significantly stronger resistance to cancer cell invasion. Comparative analysis of gene expression among mammals suggests that humans evolved higher vulnerability to malignancy than the eutherian ancestor, possibly as a correlate of more invasive placentation, and boroeutherians evolved to decrease stromal invasibility. Identifying the evolutionary determinants of stromal invasibility can provide significant insights to develop rational anti-metastatic therapeutics.

Bright near-infrared genetically encoded voltage indicator for all-optical electrophysiology

Monakhov, M. — 2019-01-31

We developed genetically encoded voltage indicators (GEVIs) using bright near-infrared (NIR) fluorescent proteins from bacterial phytochromes. These new NIR GEVIs are optimized for combination of voltage imaging with simultaneous blue light optogenetic actuator activation. Iterative optimizations led to a GEVI here termed nirButterfly, which reliably reports neuronal activities including subthreshold membrane potential depolarization and hyperpolarization, as well as spontaneous spiking, or electrically- and optogenetically-evoked action potentials. This enables largely improved all-optical causal interrogations of physiology.

SNAP23 is required for the maintenance of meiotic arrest and cortical granule exocytosis in mouse oocytes

Mehlmann, L. M. — 2019-02-28

Mammalian oocytes are stored in the ovary for prolonged periods, arrested in meiotic prophase. During this period, their plasma membranes are constantly being recycled by endocytosis and exocytosis. However, the function of this membrane turnover is unknown. Here, we investigated the requirement for exocytosis in the maintenance of meiotic arrest. Using a newly developed method for rapidly and specifically depleting proteins in oocytes, we have identified the SNARE protein, SNAP23, to be required for meiotic arrest. Degradation of SNAP23 causes premature meiotic resumption in follicle-enclosed oocytes. The reduction in SNAP23 is associated with loss of gap junction communication between the oocyte and surrounding follicle cells. Reduction of SNAP23 protein also inhibits cortical granule exocytosis in response to a Ca2+ stimulus, as measured by lectin staining and cleavage of ZP2. Our results show an essential role for SNAP23 in two key processes that occur in mouse oocytes and eggs. SummaryThe SNARE protein, SNAP23, is required to maintain gap junction communication between the oocyte and follicle cells that is needed to maintain oocyte meiotic arrest, as well as for cortical granule exocytosis at fertilization.

Macrophage mediated recognition and clearance of Borrelia burgdorferi elicits MyD88-dependent and - independent phagosomal signals that contribute to phagocytosis and inflammation

Benjamin, S. J. — 2019-03-30

Lyme disease is a tick-borne illness caused by the spirochete Borrelia burgdorferi (Bb). It is believed that the robust inflammatory response induced by the hosts innate immune system is responsible for the clinical manifestations associated with Bb infection. The macrophage plays a central role in the immune response to many bacterial infections and is thought to play a central role in activation of the innate immune response to Bb. Previous studies have shown that following phagocytosis of spirochetes by macrophages, phagosome maturation results in degradation of Bb and liberation of bacterial lipoproteins and nucleic acids, which are recognized by TLR2 and TLR8, respectively, and elicit MyD88-mediated phagosome signaling cascades. Bone marrow-derived macrophages (BMDMs) from MyD88-/- mice show significantly reduced spirochete uptake and inflammatory cytokine production when incubated with Bb ex vivo. Paradoxically, additional studies revealed that Bb-infected MyD88-/- mice exhibit inflammation in joint and heart tissues. To determine the contribution of MyD88 to macrophage-mediated spirochete clearance, we compared wildtype (WT) and MyD88-/- mice using a murine model of Lyme disease. MyD88-/- mice showed increased Bb burdens in hearts 28 days post infection, while H&E staining and immunohistochemistry showed significantly increased inflammation and greater macrophage infiltrate in the hearts of MyD88-/- mice. This suggests that Bb triggers MyD88-independent inflammatory pathways in macrophages to facilitate cell recruitment to tissues. Upon stimulation with Bb ex vivo, WT and MyD88-/- BMDMs exhibit significant differences in bacteria uptake, suggesting that MyD88 signaling mediates cytoskeleton remodeling and the formation of membrane protrusions to enhance bacteria phagocytosis. A comprehensive transcriptome comparison in Bb-infected WT and MyD88-/- BMDMs identified a large cohort of MyD88-dependent genes that are differentially expressed in response to Bb, including genes involved in actin and cytoskeleton organization (Daam1, Fmnl1). We also identified a cohort of differentially-expressed MyD88-independent chemokines (Cxcl2, Ccl9) known to recruit macrophages. We identified master regulators and generated networks which model potential signaling pathways that mediate both phagocytosis and the inflammatory response. These data provide strong evidence that MyD88-dependent and -independent phagosomal signaling cascades in macrophages play significant roles in the ability of these cells to phagocytose Bb and mediate infection.nnAUTHOR SUMMARYMacrophages play prominent roles in bacteria recognition and clearance, including Borrelia burgdorferi (Bb), the Lyme disease spirochete. To elucidate mechanisms by which MyD88/TLR signaling enhances clearance of Bb by macrophages, we studied Bb-infected wildtype (WT) and MyD88-/- mice and Bb-stimulated bone marrow-derived macrophages (BMDMs). Bb-infected MyD88-/- mice show increased bacterial burdens, macrophage infiltration and altered gene expression in inflamed heart tissue. MyD88-/- BMDMs exhibit impaired uptake of spirochetes but comparable maturation of phagosomes following internalization of spirochetes. RNA-sequencing of infected WT and MyD88-/- BMDMs identified a large cohort of differentially expressed MyD88-dependent genes involved in re-organization of actin and cytoskeleton during phagocytosis along with several MyD88-independent chemokines involved in inflammatory cell recruitment. We computationally generated networks which identified several MyD88-independent master regulators (Cxcl2 and Vcam1) and MyD88-dependent intermediate proteins (Rhoq and Cyfip1) that are known to mediate inflammation and phagocytosis respectively. These results provide mechanistic insights into MyD88-mediated phagosomal signaling enhancing Bb uptake and clearance.

Casilio-ME: Enhanced CRISPR-based DNA demethylation by RNA-guided coupling methylcytosine oxidation and DNA repair pathways

Taghbalout, A. — 2019-05-19

We have developed a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly altered the CpG methylation landscape of promoter regions and activated methylation-silenced genes. We augmented Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boosted gene activation and methylcytosine demethylation of targeted loci. We expanded the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes an advanced platform for editing DNA methylation to enable transformative research investigations interrogating DNA methylomes.

Characterization of Mucosal Dysbiosis of Early Colonic Neoplasia

Hong, B.-y. — 2019-06-06

Aberrant crypt foci (ACF) are the earliest morphologically identifiable lesion in the colon that can be detected by high-definition chromoendoscopy with contrast dye-spray. Although frequently associated with synchronous adenomas, their role in colorectal tumor development, particularly in the proximal colon, is still not clear. The goal of this study was to evaluate the profile of colon-associated bacteria associated with proximal ACF and to investigate their relationship to the presence and subtype of synchronous polyps present throughout the colon. Forty-five subjects undergoing a screening or surveillance colonoscopy were included in this retrospective study. Our study cohort included a total of 16 subjects with no identifiable proximal lesions (either ACF or polyp), 14 subjects with at least 1 ACF but no polyp(s), and 15 subjects with both at least 1 ACF and a synchronous proximal polyp(s) detected at colonoscopy. Bacterial cells adherent to the epithelia of ACF and normal mucosa were visualized by in situ hybridization within confocal sections. Bacterial DNA isolated from biopsies was used to construct PCR amplicon libraries targeting the V4 hypervariable region of the 16S rRNA gene, which were then sequenced on the Illumina platform. ACF showed significantly greater heterogeneity in their bacterial profiles compared to normal mucosa. Interestingly, one of the bacterial community structures we characterized was strongly correlated with the presence of synchronous polyps. The observed dysbiosis is more prominent within the colonic epithelium that also harbors synchronous polyps. Finally using DNA-mass spectrometry to evaluate a panel of colorectal cancer hot-spot mutations present in the ACF, we found that several APC gene mutations (R1450*, R876*, S1465fs*3) were positively associated with the presence of Instestinibacter sp., whereas KRAS mutations (G12V, G12D) were positively correlated with Ruminococcus gnavus. This result indicates a potential relationship between specific colon-associated bacterial species and somatically acquired CRC-related mutations. Overall, our findings suggest that perturbations to the normal adherent mucosal flora may constitute a risk factor for early neoplasia, demonstrating the potential impact of mucosal dysbiosis on the tissue microenvironment and behavior of ACF that may facilitate (or impede) their progression towards more advanced forms of neoplasia.

A CLEAR pipeline for direct comparison of circular and linear RNA expression

Ma, X.-K. — 2019-06-12

Sequences of circular RNAs (circRNAs) produced from back-splicing of exon(s) completely overlap with sequences from cognate linear RNAs transcribed from the same gene loci with the exception of their back-splicing junction (BSJ) sites. Examination of global circRNA expression from RNA-seq datasets generally relies on the detection of RNA-seq fragments spanning BSJ sites, but a direct comparison of circular and linear RNA expression from the same gene loci in a genome-wide manner has remained challenging. This is because quantification of BSJ fragments differs from that of linear RNA expression that uses normalized RNA-seq fragments mapped to the whole gene bodies. Here, we have developed a computational pipeline for circular and linear RNA expression analysis from ribosomal-RNA depleted RNA-seq (CLEAR, https://github.com/YangLab/CLEAR). A new quantitation parameter, FPB (fragments per billion mapped bases), is applied to evaluate circular and linear RNA expression individually by fragments mapped to circRNA-specific BSJ sites or to linear RNA-specific splicing junction (SJ) sites. Then, circular and linear RNA expression are directly compared by dividing FPBcirc by FPBlinear to generate a CIRCscore, which indicates the relative circRNA expression using linear RNA expression as the background. Highly-expressed circRNAs with low cognate linear RNA expression background can be identified for further investigation.

The HSV-1 immediate early protein ICP22 is a J-like protein required for Hsc70 reorganization during lytic infection

Adlakha, M. K. — 2019-06-14

Molecular chaperones and co-chaperones are the most abundant cellular effectors of protein homeostasis, assisting protein folding and preventing aggregation of misfolded proteins. We have previously shown that HSV-1 infection results in the drastic spatial reorganization of the cellular chaperone Hsc70 into nuclear domains called VICE (Virus Induced Chaperone Enriched) domains and that this recruitment is dependent on the viral immediate early protein ICP22. In this paper, we present several lines of evidence supporting the notion that ICP22 functions as a virally encoded co-chaperone (J-protein/Hsp40) functioning together with its Hsc70 partner to recognize and manage aggregated and misfolded proteins. We show that ICP22 results in (i) nuclear sequestration of non-native proteins, (ii) reduction of cytoplasmic aggresomes in cells expressing aggregation-prone proteins and (iii) thermoprotection against heat-inactivation of firefly luciferase. (iv) Sequence homology analysis indicated that ICP22 contains an N-terminal J-domain and a C-terminal substrate binding domain, similar to type II cellular J-proteins. ICP22 may, thus, be functionally similar to J-protein/Hsp40 co-chaperones that function together with their HSP70 partners to prevent aggregation of non-native proteins. This is not the first example of a virus hijacking a function of a cellular chaperone, as SV40 T Antigen was previously shown to contain a J-domain; however, this the first known example of the acquisition of a complete J-like protein by a virus and suggests that HSV has taken advantage of the adaptable nature of J-proteins to evolve a multi-functional co-chaperone that functions with Hsc70 to promote lytic infection.nnIMPORTANCEViruses have evolved a variety of strategies to succeed in a hostile environment. The HSV immediate early protein ICP22 plays several roles in the virus life cycle including down-regulation of cellular gene expression, up-regulation of late viral gene expression, inhibition of apoptosis, prevention of aggregation of non-native proteins and the recruitment of a cellular heat shock protein, Hsc70, to nuclear domains. We present evidence that ICP22 resembles a cellular J-protein/HSP40 family co-chaperone, interacting specifically with Hsc70. This is the first known example of the acquisition of a complete J-like protein by a virus and suggests that HSV has evolved to manipulate the host proteostatic machinery during the establishment of lytic infection.

Identifying Novel Roles for Peptidergic Signaling in Mice

Powers, K. G. — 2019-06-20

Despite accumulating evidence demonstrating the essential roles played by neuropeptides, it has proven challenging to use this information to develop therapeutic strategies. Peptidergic signaling can involve juxtacrine, paracrine, endocrine and neuronal signaling, making it difficult to define physiologically important pathways. One of the final steps in the biosynthesis of many neuropeptides requires a single enzyme, peptidylglycine -amidating monooxygenase (PAM), and lack of amidation renders most of these peptides biologically inert. PAM, an ancient integral membrane enzyme that traverses the biosynthetic and endocytic pathways, also affects cytoskeletal organization and gene expression. While mice, zebrafish and flies lacking Pam (PamKO/KO) are not viable, we reasoned that cell-type specific elimination of Pam expression would generate mice that could be screened for physiologically important and tissue-specific deficits. PamcKO/cKO mice, with loxP sites flanking the 2 exons deleted in the global PamKO/KO mouse, were indistinguishable from wildtype mice. Eliminating Pam expression in excitatory forebrain neurons reduced anxiety-like behavior, increased locomotor responsiveness to cocaine and improved thermoregulation in the cold. A number of amidated peptides play essential roles in each of these behaviors. Although atrial natriuretic peptide (ANP) is not amidated, Pam expression in the atrium exceeds levels in any other tissue. Eliminating Pam expression in cardiomyocytes increased anxiety-like behavior and improved thermoregulation. Atrial and serum levels of ANP fell sharply PamMyh6-cKO/cKO in mice and RNASeq analysis identified changes in gene expression in pathways related to cardiac function. Use of this screening platform should facilitate the development of new therapeutic approaches targeted to peptidergic pathways.nnSIGNIFICANCEPeptidergic signaling, which plays key roles in the many pathways that control thermoregulation, salt and water balance, metabolism, anxiety, pain perception and sexual reproduction, is essential for the maintenance of homeostasis. Despite the fact that peptides generally signal through G protein coupled receptors, it has proven difficult to use knowledge about peptide synthesis, storage and secretion to develop effective therapeutics. Our goal was to develop an in vivo bioassay system that would reveal physiologically meaningful deficits associated with disturbed peptidergic signaling. We did so by developing a system in which an enzyme essential for the production of many bioactive peptides could be eliminated in a tissue-specific manner.

Analysis of the three dimensional structure of the kidney glomerulus capillary network

Terasaki, M. — 2019-06-20

The capillary network of the kidney glomerulus filters small molecules from the blood. The glomerular 3D structure should help to understand its function, but it is poorly characterized. We therefore devised a new approach in which an automated tape collecting microtome(ATUM) was used to collect 0.5 micron thick serial sections from fixed mouse kidneys. The sections were imaged by scanning electron microscopy at [~]50 nm / pixel resolution. With this approach, 12 glomeruli were reconstructed at an x-y-z resolution [~]10x higher than that of paraffin sections. We found a no-cross zone between afferent and efferent branches on the vascular pole side; connections here could allow blood to exit without being adequately filtered. Network analysis indicates that the glomerular network does not form by repetitive longitudinal splitting of capillaries. It also suggests that capillaries vary their diameter to make flow more efficient. The shortest path (minimum number of branches to travel from afferent to efferent arterioles) is relatively independent of glomerular size and is present primarily on the vascular pole size. This suggests that the shortest path is established on the vascular pole side, after which new branches and longer paths form on the urinary pole side. Thus the 3D structure of the glomerular capillary network provides useful information with which to understand glomerular function. Other tissue structures in the body may benefit from this new three dimensional approach.

Control of Intracellular Molecular Networks Using Algebraic Methods

Sordo Vieira, L. — 2019-06-27

Many problems in biology and medicine have a control component. Often, the goal might be to modify intracellular networks, such as gene regulatory networks or signaling networks, in order for cells to achieve a certain phenotype, such as happens in cancer. If the network is represented by a mathematical model for which mathematical control approaches are available, such as systems of ordinary differential equations, then this problem might be solved systematically. Such approaches are available for some other model types, such as Boolean networks, where structure-based approaches have been developed, as well as stable motif techniques.nnHowever, increasingly many published discrete models are mixed-state or multistate, that is, some or all variables have more than two states, and thus the development of control strategies for multistate networks is needed. This paper presents a control approach broadly applicable to general multistate models based on encoding them as polynomial dynamical systems over a finite algebraic state set, and using computational algebra for finding appropriate intervention strategies. To demonstrate the feasibility and applicability of this method, we apply it to a recently developed multistate intracellular model of E2F-mediated bladder cancerous growth, and to a model linking intracellular iron metabolism and oncogenic pathways. The control strategies identified for these published models are novel in some cases and represent new hypotheses, or are supported by the literature in others as potential drug targets.nnOur Macaulay2 scripts to find control strategies are publicly available through GitHub at https://github.com/luissv7/multistatepdscontrol.

Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework

Brnich, S. E. — 2019-07-25

BackgroundThe American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) clinical variant interpretation guidelines established criteria (PS3/BS3) for functional assays that specified a "strong" level of evidence. However, they did not provide detailed guidance on how functional evidence should be evaluated, and differences in the application of the PS3/BS3 codes is a contributor to variant interpretation discordance between laboratories. This recommendation seeks to provide a more structured approach to the assessment of functional assays for variant interpretation and guidance on the use of various levels of strength based on assay validation.nnMethodsThe Clinical Genome Resource (ClinGen) Sequence Variant Interpretation (SVI) Working Group used curated functional evidence from ClinGen Variant Curation Expert Panel-developed rule specifications and expert opinions to refine the PS3/BS3 criteria over multiple in-person and virtual meetings. We estimated odds of pathogenicity for assays using various numbers of variant controls to determine the minimum controls required to reach moderate level evidence. Feedback from the ClinGen Steering Committee and outside experts were incorporated into the recommendations at multiple stages of development.nnResultsThe SVI Working Group developed recommendations for evaluators regarding the assessment of the clinical validity of functional data and a four-step provisional framework to determine the appropriate strength of evidence that can be applied in clinical variant interpretation. These steps are: 1. Define the disease mechanism; 2. Evaluate applicability of general classes of assays used in the field; 3. Evaluate validity of specific instances of assays; 4. Apply evidence to individual variant interpretation. We found that a minimum of eleven total pathogenic and benign variant controls are required to reach moderate-level evidence in the absence of rigorous statistical analysis.nnConclusionsThe recommendations and approach to functional evidence evaluation described here should help clarify the clinical variant interpretation process for functional assays. Further, we hope that these recommendations will help develop productive partnerships with basic scientists who have developed functional assays that are useful for interrogating the function of a variety of genes.

Molecular correlates of topiramate and GRIK1 rs2832407 genotype in pluripotent stem cell-derived neural cultures

Lieberman, R. — 2019-07-22

There is growing evidence that the anticonvulsant topiramate is efficacious in reducing alcohol consumption. Further, an intronic single nucleotide polymorphism (rs2832407, C A) in the GRIK1 gene, which encodes the GluK1 subunit of the excitatory kainate receptor, predicted topiramates effectiveness in reducing heavy drinking in a clinical trial. In the current study, we differentiated a total of 22 induced pluripotent stem cell (iPSCs) lines characterized by GRIK1 rs2832407 genotype (10 A/A and 12 C/C) into forebrain-lineage neural cultures to explore molecular correlates of GRIK1 genotype that may relate to topiramates ability to reduce drinking. Our differentiation protocol yielded mixed neural cultures enriched for glutamatergic neurons. Characterization of the GRIK1 locus revealed no effect of rs2832407 genotype on GRIK1 isoform mRNA expression, however a significant difference was observed on GRIK1 antisense-2, with higher expression in C/C neural cultures. Differential effects of acute exposure to 5 M topiramate were observed on the frequency of spontaneous synaptic activity in A/A vs. C/C neurons, with a smaller reduction in excitatory event frequency and a greater reduction in inhibitory event frequency observed in C/C donor neurons. This work highlights the use of iPSC technologies to study pharmacogenetic treatment effects in psychiatric disorders and furthers our understanding of the molecular effects of topiramate exposure in human neural cells.

Shared Genetic Risk between Eating Disorder- and Substance-Use-Related Phenotypes: Evidence from Genome-Wide Association Studies

Munn-Chernoff, M. — 2019-08-23

Eating disorders and substance use disorders frequently co-occur. Twin studies reveal shared genetic variance between liabilities to eating disorders and substance use, with the strongest associations between symptoms of bulimia nervosa (BN) and problem alcohol use (genetic correlation [rg], twin-based=0.23-0.53). We estimated the genetic correlation between eating disorder and substance use and disorder phenotypes using data from genome-wide association studies (GWAS). Four eating disorder phenotypes (anorexia nervosa [AN], AN with binge-eating, AN without binge-eating, and a BN factor score), and eight substance-use-related phenotypes (drinks per week, alcohol use disorder [AUD], smoking initiation, current smoking, cigarettes per day, nicotine dependence, cannabis initiation, and cannabis use disorder) from eight studies were included. Significant genetic correlations were adjusted for variants associated with major depressive disorder (MDD). Total sample sizes per phenotype ranged from ~2,400 to ~537,000 individuals. We used linkage disequilibrium score regression to calculate single nucleotide polymorphism-based genetic correlations between eating disorder and substance-use-related phenotypes. Significant positive genetic associations emerged between AUD and AN (rg=0.18; false discovery rate q=0.0006), cannabis initiation and AN (rg=0.23; q<0.0001), and cannabis initiation and AN with binge-eating (rg=0.27; q=0.0016). Conversely, significant negative genetic correlations were observed between three non-diagnostic smoking phenotypes (smoking initiation, current smoking, and cigarettes per day) and AN without binge-eating (rgs=-0.19 to -0.23; qs<0.04). The genetic correlation between AUD and AN was no longer significant after co-varying for MDD loci. The patterns of association between eating disorder- and substance-use-related phenotypes highlights the potentially complex and substance-specific relationships between these behaviors.

Transcriptional profiling of non-injured nociceptors after spinal cord injury reveals diverse molecular changes

Yasko, J. R. — 2019-08-22

Traumatic spinal cord injury (SCI) has devastating implications for patients, including a high predisposition for developing chronic pain distal to the site of injury. Chronic pain develops weeks to months after injury, consequently patients are treated after irreparable changes have occurred. Nociceptors are central to chronic pain; however, the diversity of this cellular population presents challenges to understanding mechanisms and attributing pain modalities to specific cell types. To begin to address how peripheral sensory neurons distal to the site of injury may contribute to the below-level pain reported by SCI patients, we examined SCI-induced changes in gene expression in lumbar dorsal root ganglia (DRG) below the site of injury. SCI was performed at the T10 vertebral level, with injury produced by a vessel clip with a closing pressure of 15g for 1 minute. Alterations in gene expression produce long-term sensory changes, therefore we were interested in studying SCI-induced transcripts before the onset of chronic pain, which may trigger changes in downstream signaling pathways and ultimately facilitate the transmission of pain. To examine changes in the nociceptor subpopulation in DRG distal to the site of injury, we retrograde labeled sensory neurons projecting to the hairy hindpaw skin with fluorescent dye and collected the corresponding lumbar (L2-L6) DRG 4 days post-injury. Following dissociation, labeled neurons were purified by fluorescence-activated cell sorting. RNA was extracted from sorted sensory neurons of naive, sham, or SCI mice and sequenced. Transcript abundances validated that the desired population of nociceptors were isolated. Cross-comparisons to data sets from similar studies confirmed we were able to isolate our cells of interest and identify a unique pattern of gene expression within a subpopulation of neurons projecting to the hairy hindpaw skin. Differential gene expression analysis showed high expression levels and significant transcript changes 4 days post-injury in SCI cell populations relevant to the onset of chronic pain. Regulatory interrelationships predicted by pathway analysis implicated changes within the synaptogenesis signaling pathway as well as networks related to inflammatory signaling mechanisms, suggesting a role for synaptic plasticity and a correlation with pro-inflammatory signaling in the transition from acute to chronic pain.nnContribution to the fieldTraumatic spinal cord injury (SCI) has devastating implications for patients, including a high predisposition for developing chronic pain. Much of the pain seems to emanate from tissues further away from the brain than the site of injury. Chronic pain develops weeks to months after injury, which means that patients are frequently treated only after enduring pain has developed. Nociceptors are the specialized sensory neurons central to chronic pain. We were interested in studying SCI-induced gene transcript (RNA) changes before the onset of chronic pain, in the hope of identifying mechanisms which could become therapeutic targets. Nociceptors below the site of spinal injury were isolated and their RNAs were sequenced. The results identified a unique pattern of gene expression in the subpopulation of nociceptors projecting to the relevant peripheral tissue. Particularly interesting were sets of genes crucial to synapse formation and maturation - the ability of neurons to talk to each other - and genes involved in inflammatory responses, since treatment of inflammation of nervous tissue could also be important for therapeutic approaches. It is evident that the transition from acute to chronic pain occurs in distinct steps that involve numerous signaling pathways, providing a host of potential new drug targets.

Dissecting the Roles of Kalirin-7/PSD95/GluN2B Interactions in Different Forms of Synaptic Plasticity

Yeh, M. L. — 2019-08-22

Kalirin-7 (Kal7) is a Rac1/RhoG GEF and multidomain scaffold localized to the postsynaptic density which plays an important role in synaptic plasticity. Behavioral and physiological phenotypes observed in the Kal7 knockout mouse are quite specific: genetics of breeding, growth, strength and coordination are normal; Kal7 knockout animals self-administer cocaine far more than normal mice, show exaggerated locomotor responses to cocaine, but lack changes in dendritic spine morphology seen in wildtype mice; Kal7 knockout mice have depressed surface expression of GluN2B receptor subunits and exhibit marked suppression of long-term potentiation and depression in hippocampus, cerebral cortex, and spinal cord; and Kal7 knockout mice have dramatically blunted perception of pain. To address the underlying cellular and molecular mechanisms which are deranged by loss of Kal7, we administered intracellular blocking peptides to acutely change Kal7 function at the synapse, to determine if plasticity deficits in Kal7-/-mice are the product of developmental processes since conception, or could be detected on a much shorter time scale. We found that specific disruption of the interactions of Kal7 with PSD-95 or GluN2B resulted in significant suppression of long-term potentiation and long-term depression. Biochemical approaches indicated that Kal7 interacted with PSD-95 at multiple sites within Kal7.nnGraphical Table of ContentsThe postsynaptic density is an integral player in receiving, interpreting and storing signals transmitted by presynaptic terminals. The correct molecular composition is crucial for successful expression of synaptic plasticity. Key components of the postsynaptic density include ligand-gated ion channels, structural and binding proteins, and multidomain scaffolding plus enzymatic proteins. These studies address whether the multiple components of the synaptic density bind together in a static or slowly adapting molecular complex, or whether critical interactions are fluid on a minute-to-minute basis.nnnnO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/744508v1_ufig1.gif" ALT="Figure 1">nView larger version (55K):norg.highwire.dtl.DTLVardef@a65aeborg.highwire.dtl.DTLVardef@19b175borg.highwire.dtl.DTLVardef@11a70ceorg.highwire.dtl.DTLVardef@e403b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Polygenic Prediction of Substance Use Disorders in Clinical and Population Samples

Barr, P. B. — 2019-08-30

Genome-wide, polygenic risk scores (PRS) have emerged as a useful way to characterize genetic liability using genotypic data. There is growing evidence that PRS may prove useful to identify those at increased risk for developing certain diseases. The current utility of PRS in relation to alcohol use disorders (AUD) remains an open question. Using data from both a population-based sample [the FinnTwin12 (FT12) study] and a high risk sample [the Collaborative Study on the Genetics of Alcoholism (COGA)], we examined the association between PRSs derived from genome-wide association studies (GWASs) of 1) alcohol dependence/alcohol problems, 2) alcohol consumption, and 3) risky behaviors with AUD and other substance use disorder (SUD) symptoms. Individuals in the top 20%, 10%, and 5% of PRSs had increasingly greater odds of having an AUD compared to the lower end of the continuum in both COGA (80th % OR = 1.95; 90th % OR = 2.03; 95th % OR = 2.13) and FT12 (80th % OR = 1.77; 90th % OR = 2.27; 95th % OR = 2.39). Those in the top 5% reported greater levels of licit (alcohol and nicotine) and illicit (cannabis) SUD symptoms. PRSs can predict elevated risk for SUD in independent samples. However, clinical utility of these scores in their current form is modest. As these scores become more predictive of SUD, they may become useful to practitioners. Improvement in predictive ability will likely be dependent on increasing the size of well-phenotyped discovery samples.

Sensitivity and robustness of comorbidity network analysis

Brunson, J. C. — 2019-09-05

1Summary and KeywordsO_ST_ABSBackgroundC_ST_ABSComorbidity network analysis (CNA) is an increasingly popular approach in systems medicine, in which mathematical graphs encode epidemiological correlations (links) between diseases (nodes) inferred from their occurrence in an underlying patient population. A variety of methods have been used to infer properties of the constituent diseases or underlying populations from the network structure, but few have been validated or reproduced.nnObjectivesTo test the robustness and sensitivity of several common CNA techniques to the source of population health data and the method of link determination.nnMethodsWe obtained six sources of aggregated disease co-occurrence data, coded using varied ontologies, most of which were provided by the authors of CNAs. We constructed families of comorbidity networks from these data sets, in which links were determined using a range of statistical thresholds and measures of association. We calculated degree distributions, single-value statistics, and centrality rankings for these networks and evaluated their sensitivity to the source of data and link determination parameters. From two open-access sources of patient-level data, we constructed comorbidity networks using several multivariate models in addition to comparable pairwise models and evaluated differences between correlation estimates and network structure.nnResultsGlobal network statistics vary widely depending on the underlying population. Much of this variation is due to network density, which for our six data sets ranged over three orders of magnitude. The statistical threshold for link determination also had strong effects on global statistics, though at any fixed threshold the same patterns distinguished our six populations. The association measure used to quantify comorbid relations had smaller but discernible effects on global structure. Co-occurrence rates estimated using multivariate models were increasingly negative-shifted as models accounted for more effects. However, only associations between the most prevalent disorders were consistent from model to model. Centrality rankings were likewise similar when based on the same dataset using different constructions; but they were difficult to compare, and very different when comparable, between data sets, especially those using different ontologies. The most central disease codes were particular to the underlying populations and were often broad categories, injuries, or non-specific symptoms.nnConclusionsCNAs can improve robustness and comparability by accounting for known limitations. In particular, we urge comorbidity network analysts (a) to include, where permissible, disaggregated disease occurrence data to allow more targeted reproduction and comparison of results; (b) to report differences in results obtained using different association measures, including both one of relative risk and one of correlation; (c) when identifying centrally located disorders, to carefully decide the most suitable ontology for this purpose; and, (d) when relevant to the interpretation of results, to compare them to those obtained using a multivariate model.

Targeting the Pregnane X Receptor Using Microbial Metabolite Mimicry

Dvorak, Z. — 2019-10-04

The human pregnane X receptor (PXR), a master regulator of drug metabolism, has important roles in intestinal homeostasis and abrogating inflammation. Existing PXR ligands have substantial off-target toxicity. Based on prior work that established microbial (indole) metabolites as PXR ligands, we proposed microbial metabolite mimicry as a novel strategy for drug discovery that allows to exploit previously unexplored parts of chemical space. Here we report functionalized indole-derivatives as first-in-class non-cytotoxic PXR agonists, as a proof-of-concept for microbial metabolite mimicry. The lead compound, FKK6, binds directly to PXR protein in solution, induces PXR specific target gene expression in, cells, human organoids, and mice. FKK6 significantly represses pro-inflammatory cytokine production cells and abrogates inflammation in mice expressing the human PXR gene. The development of FKK6 demonstrates for the first time that microbial metabolite mimicry is a viable strategy for drug discovery and opens the door to mine underexploited regions of chemical space.

Mad dephosphorylation at the nuclear envelope is essential for asymmetric stem cell division

Sardi, J. — 2019-10-16

Stem cell niche signals act over a short range so that only stem cells but not the differentiating daughter cells receive the self-renewal signals. Drosophila female germline stem cells (GSCs) are maintained by short range BMP signaling; BMP ligands Dpp/Gbb activate receptor Tkv to phosphorylate Mad (phosphor-Mad or pMad) which accumulates in the GSC nucleus and activates the stem cell transcription program. pMad is highly concentrated in the nucleus of the GSC, but is immediately downregulated in the nucleus of the pre-cystoblast (preCB), a differentiating daughter cell, that is displaced away from the niche. Here we show that this asymmetry in the intensity of pMad is formed even before the completion of cytokinesis. A delay in establishing the pMad asymmetry leads to germline tumors through conversion of differentiating cells into a stem cell-like state. We show that a Mad phosphatase Dullard (Dd) interacts with Mad at the nuclear pore, where it may dephosphorylate Mad. A mathematical model explains how an asymmetry can be established in a common cytoplasm. It also demonstrates that the ratio of pMad concentrations in GSC/preCB is highly sensitive to Mad dephosphorylation rate. Our study reveals a previously unappreciated mechanism for breaking symmetry between daughter cells during asymmetric stem cell division.

OCSANA+: Optimal Control and Simulation of Signaling Networks from Network Analysis

Marazzi, L. — 2019-10-16

SummaryOCSANA+ is a Cytoscape app for identifying nodes to drive the system towards a desired long-term behavior, prioritizing combinations of interventions in large scale complex networks, and estimating the effects of node perturbations in signaling networks, all based on the analysis of the networks structure. OCSANA+ includes an update to OCSANA (optimal combinations of interventions from network analysis) software tool with cutting-edge and rigorously tested algorithms, together with recently-developed structure-based control algorithms for non-linear systems and an algorithm for estimating signal flow. All these algorithms are based on the networks topology. OCSANA+ is implemented as a Cytoscape app to enable a user interface for running analyses and visualizing results. Availability and ImplementationOCSANA+ app and its tutorial can be downloaded from the Cytoscape App Store or https://veraliconaresearchgroup.github.io/OCSANA-Plus/. The source code and computations are available in https://github.com/VeraLiconaResearchGroup/OCSANA-Plus_SourceCode.

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins.

Van Nostrand, E. L. — 2019-10-16

A critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs). Crosslinking and immunoprecipitation (CLIP) methods enabled mapping RBP targets transcriptome-wide, but methodological differences present challenges to large-scale integrated analysis across datasets. The development of enhanced CLIP (eCLIP) enabled the large-scale mapping of targets for 150 RBPs in K562 and HepG2, creating a unique resource of RBP interactomes profiled with a standardized methodology in the same cell types. Here we describe our analysis of 223 enhanced (eCLIP) datasets characterizing 150 RBPs in K562 and HepG2 cell lines, revealing a range of binding modalities, including highly resolved positioning around splicing signals and mRNA untranslated regions that associate with distinct RBP functions. Quantification of enrichment for repetitive and abundant multi-copy elements reveals 70% of RBPs have enrichment for non-mRNA element classes, enables identification of novel ribosomal RNA processing factors and sites and suggests that association with retrotransposable elements reflects multiple RBP mechanisms of action. Analysis of spliceosomal RBPs indicates that eCLIP resolves AQR association after intronic lariat formation (enabling identification of branch points with single-nucleotide resolution) and provides genome-wide validation for a branch point-based scanning model for 3 splice site recognition. Further, we show that eCLIP peak co-occurrences across RBPs enables the discovery of novel co-interacting RBPs. Finally, we present a protocol for visualization of RBP:RNA complexes in the eCLIP workflow using biotin and standard chemiluminescent visualization reagents, enabling simplified confirmation of ribonucleoprotein enrichment without radioactivity. This work illustrates the value of integrated analysis across eCLIP profiling of RBPs with widely distinct functions to reveal novel RNA biology. Further, our quantification of both mRNA and other element association will enable further research to identify novel roles of RBPs in regulating RNA processing.

ApoC-III overexpression and LDLr-/- protect mice from DSS-colitis: identifying a new role for lipoprotein metabolism in Tregs

Rodia, C. N. — 2019-11-05

ObjectiveCellular metabolism is a key regulator of CD4+Foxp3+ regulatory T cell (Treg) homeostasis, but the foundational studies in this area use free fatty acid treatment as a proxy for plasma triglycerides. In vivo, plasma triglyceride is the main source of fatty acids for cells, not free fatty acids. Design/ResultsUsing apolipoprotein C-III transgenic and LDLr-/- mice, we report that the loss of lipoprotein triglycerides transport in these models results in protection from DSS-colitis and accumulation of intestinal Tregs and plasma IL-10. Total loss of apoC-III increases colitis severity. Tregs exposed to apoC-III increase lipolysis and fatty acid oxidation and apoC-III inhibits Bodipy-triglyceride uptake. Therapeutic treatment of WT mice with apoC-III-containing lipoproteins protects mice from colitis. ConclusionOur data suggest that therapies that reduce apoC-III could have negative effects in patients who are at risk of IBD, and conversely, that apoC-III could be a new therapeutic target to stimulate intestinal Tregs and IL-10 for the management of IBD. These data identify apoC-III and lipoprotein metabolism as a novel regulator of tolerance in the intestine. Summary Box* What is already known about this subject:{blacksquare} The relative capacity to use either glucose or FFA to generate acetyl CoA for mitochondrial fatty acid oxidation is a critical driver of Treg and T cell activity and proliferation. {blacksquare}ApoC-III is a known regulator of triglyceride and fatty acid metabolism in cells via LPL and LDLr endocytosis pathways {blacksquare}ApoC-III is reduced in Crohns and Colitis patients. * What are the new findings:{blacksquare} We show that Tregs express triglyceride transporters, and that LDLr expression is enriched in Tregs from the mesenteric lymph nodes. {blacksquare}We show that T cells are capable of endocytosing triglyceride from lipoproteins, and this process is inhibited by apoC-III. {blacksquare}Tregs from apoC-IIITg are metabolically unique from WT Tregs and they upregulate the genes of lipolysis, and have an increase in basal respiration. {blacksquare}The inhibition of TAG endocytosis, using 2 different models (LDLrKO and apoC-III-transgenic mice), protects mice from colitis and stimulates the accumulation of Tregs and IL-10 in the gut. {blacksquare}Intraperitoneal delivery of apoC-III on chylomicrons protects WT mice from DSS colitis. * How might it impact on clinical practice in the foreseeable future?{blacksquare} Due to the protective role apoC-III plays in these mouse models of colitis, IBD risk should be carefully considered before prescribing patient anti-apoC-III lipid-lowering therapies.

Local Glutamate-Mediated Dendritic Plateau Potentials Change the State of the Cortical Pyramidal Neuron

Gao, P. P. — 2019-11-02

Dendritic spikes in thin dendritic branches (basal and oblique dendrites) of pyramidal neurons are traditionally inferred from spikelets measured in the cell body. Here, we used laser-spot voltage-sensitive dye imaging in cortical pyramidal neurons (rat brain slices) to investigate the voltage waveforms of dendritic potentials occurring in response to spatially-restricted glutamatergic inputs. Local dendritic potentials lasted 200-500 ms and propagated to the cell body where they caused sustained 10-20 mV depolarizations. Plateau potentials propagating from dendrite to soma, and action potentials propagating from soma to dendrite, created complex voltage waveforms in the middle of the thin basal dendrite, comprised of local sodium spikelets, local plateau potentials, and back-propagating action potentials, superimposed on each other. Our model replicated these experimental observations and made predictions, which were tested in experiments. Dendritic plateau potentials occurring in basal and oblique branches put pyramidal neurons into an activated neuronal state ("prepared state"), characterized by depolarized membrane potential and faster membrane responses. The prepared state provides a time window of 200-500 ms during which cortical neurons are particularly excitable and capable of following afferent inputs. At the network level, this predicts that sets of cells with simultaneous plateaus would provide cellular substrate for the formation of functional neuronal ensembles. New & NoteworthyIn cortical pyramidal neurons, we recorded glutamate-mediated dendritic plateau potentials using voltage imaging, and created a computer model that recreated experimental measures from dendrite and cell body. Our model made new predictions, which were then tested in experiments. Plateau potentials profoundly change neuronal state -- a plateau potential triggered in one basal dendrite depolarizes the soma and shortens membrane time constant, making the cell more susceptible to firing triggered by other afferent inputs.

Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

Werneburg, S. — 2019-11-15

Multiple sclerosis (MS) is a demyelinating, autoimmune disease of the central nervous system. While work has focused on axon loss in MS, far less is known about synaptic changes. Here, in striking similarity to other neurodegenerative diseases, we identify in postmortem human MS tissue and in nonhuman primate and mouse MS models profound synapse loss and microglial synaptic engulfment. These events can occur independently of local demyelination, neuronal degeneration, and peripheral immune cell infiltration, but coincide with gliosis and increased localization of complement component C3, but not C1q, at synapses. Finally, we use AAV9 to overexpress the complement inhibitor Crry at activated C3-bound synapses in mice and demonstrate robust protection of synapses and visual function. These results mechanistically dissect synapse loss as an early pathology in MS. We further provide a novel gene therapy approach to prevent synapse loss by microglia, which may be broadly applicable to other neurodegenerative diseases.

Massively parallel disruption of enhancers active during human corticogenesis

Geller, E. — 2019-12-02

Changes in gene regulation have been linked to the expansion of the human cerebral cortex and to neurodevelopmental disorders. However, the biological effects of genetic variation within developmental regulatory elements on human corticogenesis are not well understood. We used sgRNA-Cas9 genetic screens in human neural stem cells (hNSCs) to disrupt 10,674 expressed genes and 2,227 enhancers active in the developing human cortex and determine the resulting effects on cellular proliferation. Gene disruptions affecting proliferation were enriched for genes associated with risk for human neurodevelopmental phenotypes including primary microcephaly and autism spectrum disorder. Although disruptions in enhancers had overall weaker effects on proliferation than gene disruptions, we identified enhancer disruptions that severely perturbed hNSC self-renewal. Disruptions in Human Accelerated Regions and Human Gain Enhancers, regulatory elements implicated in the evolution of the human brain, also perturbed hNSC proliferation, establishing a role for these elements in human neurodevelopment. Integrating proliferation phenotypes with chromatin interaction maps revealed regulatory relationships between enhancers and target genes that contribute to neurogenesis and potentially to human cortical evolution.

Slo2 potassium channel function depends on a SCYL1 protein

Niu, L.-G. — 2019-12-04

Slo2 potassium channels play important roles in neuronal function, and their mutations in humans cause epilepsies and cognitive defects. However, little is known how Slo2 function is regulated by other proteins. Here we found that the function of C. elegans Slo2 (SLO-2) depends on adr-1, a gene important to RNA editing. However, slo-2 transcripts have no detectable RNA editing events and exhibit similar expression levels in wild type and adr-1 mutants. In contrast, mRNA level of scyl-1, which encodes an orthologue of mammalian SCYL1, is greatly reduced in adr-1 mutants due to deficient RNA editing at a single adenosine in its 3-UTR. SCYL-1 physically interacts with SLO-2 in neurons. Single-channel open probability of SLO-2 in neurons is reduced by [~]50% in scyl-1 knockout whereas that of human Slo2.2/Slack is doubled by SCYL1 in a heterologous expression system. These results suggest that SCYL-1/SCYL1 is an evolutionarily conserved regulator of Slo2 channels.

Massively parallel discovery of human-specific substitutions that alter neurodevelopmental enhancer activity

Uebbing, S. — 2019-12-05

Genetic changes that altered the function of gene regulatory elements have been implicated in the evolution of the human brain. However, identifying the particular changes that modified regulatory activity during neurodevelopment remains challenging. Here we used massively parallel enhancer assays in human neural stem cells to measure the impact of 32,776 human-specific substitutions on enhancer activity in 1,363 Human Accelerated Regions (HARs) and 3,027 Human Gain Enhancers (HGEs), which include enhancers with novel activities in humans. We found that 31.9% of active HARs and 36.4% of active HGEs exhibited differential activity between human and chimpanzee. This enabled us to isolate the effects of 401 human-specific substitutions from other types of genetic variation in HARs and HGEs. Substitutions acted in both an additive and non-additive manner to alter enhancer activity. Human-specific substitutions altered predicted binding sites for a specific set of human transcription factors (TFs) that were a subset of TF binding sites associated with enhancer activity in our assay. Substitutions within HARs, which are overall highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions may be buffered in enhancers with constrained ancestral functions. Our findings yield insight into the mechanisms by which human-specific genetic changes impact enhancer function and provide a rich set of candidates for experimental studies of regulatory evolution in humans.

Dkk2 interacts with Pax9 in palate mesenchyme to pattern and tune osteogenesis

Oliver Pina, J. — 2023-05-17

Multiple genetic and environmental etiologies contribute to the pathogenesis of cleft palate, which constitutes the most common among the inherited disorders of the craniofacial complex. Insights into the molecular mechanisms regulating osteogenic differentiation and patterning in the palate during embryogenesis are limited and needed for the development of innovative diagnostics and cures. This study utilized the Pax9-/- mouse model with a consistent phenotype of cleft secondary palate to investigate the role of Pax9 in the process of palatal osteogenesis. While prior research had identified upregulation of Wnt pathway modulators Dkk1 and Dkk2 in Pax9-/- palate mesenchyme, limitations of spatial resolution and technology restricted a more robust analysis. Here, data from single-nucleus transcriptomics and chromatin accessibility assays validated by in situ highly multiplex targeted single-cell spatial profiling technology suggest a distinct relationship between Pax9+ and osteogenic populations. Loss of Pax9 results in spatially restricted osteogenic domains bounded by Dkk2, which normally interfaces with Pax9 in the mesenchyme. These results suggest that Pax9-dependent Wnt signaling modulators influence osteogenic programming during palate formation, potentially contributing to the observed cleft palate phenotype.

Phosphatases modified by LH signaling in rat ovarian follicles and their role in regulating the NPR2 guanylyl cyclase

Egbert, J. R. — 2023-06-12

In response to luteinizing hormone, multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-minute exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the PPP family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4-10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/544636v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@2622cdorg.highwire.dtl.DTLVardef@1b6eef7org.highwire.dtl.DTLVardef@102da9borg.highwire.dtl.DTLVardef@5e3221_HPS_FORMAT_FIGEXP M_FIG C_FIG Summary sentenceQuantitative mass spectrometric analysis of phosphatases whose phosphorylation state is rapidly modified by luteinizing hormone provides clues about how LH signaling dephosphorylates NPR2 as well as a resource for future studies.

Borrelia PeptideAtlas: A proteome resource of common Borrelia burgdorferi isolates for Lyme research

Reddy, P. J. — 2023-06-16

Lyme disease, caused by an infection with the spirochete Borrelia burgdorferi, is the most common vector-borne disease in North America. B. burgdorferi strains harbor extensive genomic and proteomic variability and further comparison is key to understanding the spirochetes infectivity and biological impacts of identified sequence variants. To achieve this goal, both transcript and mass spectrometry (MS)-based proteomics was applied to assemble peptide datasets of laboratory strains B31, MM1, B31-ML23, infective isolates B31-5A4, B31-A3, and 297, and other public datasets, to provide a publicly available Borrelia PeptideAtlas (http://www.peptideatlas.org/builds/borrelia/). Included is information on total proteome, secretome, and membrane proteome of these B. burgdorferi strains. Proteomic data collected from 35 different experiment datasets, with a total of 855 mass spectrometry runs, identified 76,936 distinct peptides at a 0.1% peptide false-discovery-rate, which map to 1,221 canonical proteins (924 core canonical and 297 noncore canonical) and covers 86% of the total base B31 proteome. The diverse proteomic information from multiple isolates with credible data presented by the Borrelia PeptideAtlas can be useful to pinpoint potential protein targets which are common to infective isolates and may be key in the infection process.

Comprehensive analysis of network reconstruction approaches based on correlation in metagenomic data

Fuschi, A. — 2023-06-22

Microbiome analysis is transforming our understanding of biological processes related to human health, epidemiology (antimicrobial resistance, horizontal gene transfer) environmental and agricultural studies. At the core of microbiome analysis is the description of microbial communities based on quantification of microbial taxa and dynamics. In the study of bacterial abundances, it is becoming more relevant to consider their relationship, to embed these data in the framework of network theory, allowing characterization of features like node relevance, pathway and community structure. In this work we characterize the principal biases in reconstructing networks from correlation measures, associated with the compositional character of relative abundance data, the diversity of abundances and the presence of unobserved species within a single sample, that might lead to wrong correlation estimates. We show how most of these problems can be overcome by applying typical transformations for compositional data, that allow the application of simple measures such as Pearsons correlation to correctly identify the positive and negative relationships between relative abundances, when data dimensionality is sufficiently high. Some issues remain, like the role of data sparsity, that if not properly addressed can lead to imbalances in correlation coefficient distribution.

Zona incerta distributes a broad movement signal that modulates behavior

Hormigo, S. — 2023-06-22

The zona incerta is a subthalamic nucleus made up mostly of GABAergic neurons. It has wide- ranging inputs and outputs and is believed to have many integrative functions that link sensory stimuli with motor responses to guide behavior. However, its role is not well established perhaps because few studies have measured the activity of zona incerta neurons in behaving animals under different conditions. To record the activity of zona incerta neurons during exploratory and cue-driven goal-directed behaviors, we used electrophysiology in head-fixed mice moving on a spherical treadmill and fiber photometry in freely moving mice. We found two groups of neurons based on their sensitivity to movement, with a minority of neurons responding to whisker stimuli. Furthermore, zona incerta GABAergic neurons robustly code the occurrence of exploratory and goal-directed movements, but not their direction. To understand the function of these activations, we performed genetically targeted lesions and optogenetic manipulations of zona incerta GABAergic neurons during exploratory and goal-directed behaviors. The results showed that the zona incerta has a role in modulating the movement associated with these behaviors, but this has little impact on overall performance. Zona incerta neurons distribute a broad corollary signal of movement occurrence to their diverse projection sites, which regulates behavior.

ER-Mitochondria Contact Sites expand during mitosis

Yu, F. — 2023-06-22

Membrane contact sites between various organelles define specialized spatially defined signaling hubs, they are of great interest to better understand inter-organelle communication and its implications on cellular physiology. ER-mitochondria contact sites (ERMCS) are one of the best studied and mediate Ca2+ signaling that regulates mitochondrial bioenergetics. However, little is known about ERMCS during mitosis. Here we show that ERMCS expand during mitosis using transmission electron microscopy, serial EM coupled to 3D reconstruction, and ERMCS markers. ERMCS expansion in mitosis is functionally significant as it is associated with enhanced Ca2+ coupling between the ER and mitochondria resulting in heightened activation of mitochondrial dehydrogenases. Our data suggest that ERMCS remodeling in mitosis is important to meet the increased energy needs during cell division.

Histone deacetylase 3 plays a critical role in hypertension-induced kidney inflammation and fibrosis

An, C. — 2023-06-29

Objective: Hypertension is a major cause of chronic kidney disease. The molecular mechanisms how hypertension causes kidney disease are not fully understood. In this study, we examined the functional role of histone deacetylase 3 (HDAC3) in the pathogenesis of hypertensive kidney disease. Approach and Results: Myeloid HDAC3 knockout mice were generated by crossing HDAC3f/f mice with LysM-Cre+/- mice. Both LysM-Cre-/-HDAC3f/f (HDAC3CON) mice and LysM-Cre+/+HDAC3f/f (HDAC3MKO) mice were subjected to uninephrectomy and treated with angiotensin II at 1.5 ug/kg/min or vehicle for 28 days. Blood pressure increased comparably in both HDAC3CON and HDAC3MKO mice following angiotensin II treatment. Compared with HDAC3CON mice, HDAC3MKO mice were protected from angiotensin II-induced kidney dysfunction and pathological injury and developed less proteinuria. Myeloid HDAC3 deficiency markedly attenuated the protein levels of fibronectin, collagen I, and ?-SMA in the kidney following angiotensin II treatment. Furthermore, HDAC3MKO mice accumulated significantly fewer myeloid fibroblasts and macrophages in the kidney after angiotensin II treatment. Myeloid HDAC3 deficiency significantly reduced the mRNA expression of proinflammatory molecules in the kidney following angiotensin II treatment. In cultured macrophages, knockdown of HDAC3 with shRNA suppressed proinflammatory molecule expression. Furthermore, knockdown of HDAC3 inhibited NF-kB p65 enrichment to IL-6 promoter. Conclusion: Our study identifies a critical role of myeloid HDAC3 in the regulation of hypertension-induced kidney inflammation and fibrosis. Targeting HDAC3 may represent a novel therapeutic strategy for hypertensive kidney disease.

TRPS1 modulates chromatin accessibility to regulate estrogen receptor (ER) binding and ER target gene expression in luminal breast cancer cells

Scott, T. G. — 2023-07-03

Breast cancer is the most frequently diagnosed cancer in women. The most common subtype is luminal breast cancer, which is typically driven by the estrogen receptor (ER), a transcription factor (TF) that activates many genes required for proliferation. Multiple effective therapies target this path-way, but individuals often develop resistance. Thus, there is a need to identify additional targets that regulate ER activity and contribute to breast tumor progression. TRPS1 is a repressive GATA-family TF that is overexpressed in breast tumors. Common genetic variants in the TRPS1 locus are associated with breast cancer risk, and luminal breast cancer cell lines are particularly sensitive to TRPS1 knockout. However, we do not know how TRPS1 regulates target genes to mediate these breast cancer patient and cellular outcomes. We introduced an inducible degron tag into the native TRPS1 locus within a luminal breast cancer cell line to identify the direct targets of TRPS1 and determine how TRPS1 mechanistically regulates gene expression. We acutely deplete over eighty percent of TRPS1 from chromatin within 30 minutes of inducing degradation. We find that TRPS1 regulates transcription of hundreds of genes, including those related to estrogen signaling. TRPS1 directly regulates chromatin structure, which causes ER to redistribute in the genome. ER redistribution leads to both repression and activation of dozens of ER target genes. Downstream from these primary effects, TRPS1 depletion represses cell cycle-related gene sets and reduces cell doubling rate. Finally, we show that high TRPS1 activity, calculated using a gene expression signature defined by primary TRPS1-regulated genes, is associated with worse breast cancer patient prognosis. Taken together, these data suggest a model in which TRPS1 modulates the activity of other TFs, both activating and repressing transcription of genes related to cancer cell fitness.

Experimental and Phylogenetic Evidence for Correlated Gene Expression Evolution between Dermal and Endometrial Fibroblasts: implications for the evolution of cancer malignancy

Dighe, A. — 2023-07-09

Changes in transcriptional gene expression is a dominant mode of evolution, mostly driven by mutations at cis-regulatory regions. Mutations can affect gene expression in multiple cell types if the same cis-regulatory elements are used by different cell types. As a consequence, changes in gene expression in one cell type may be associated with similar gene expression changes in another cell type. Correlated gene expression change can explain correlated character evolution, as for instance the correlation between placental invasion and vulnerability to cancer malignancy. Here we test this hypothesis using a comparative and an experimental data set. Specifically, we investigate gene expression in dermal skin fibroblasts (SF) and uterine endometrial stomal fibroblasts (ESF). The comparative dataset consists of transcriptomes from cultured SF and ESF from 9 mammalian species. We calculated the independent phylogenetic contrasts (PIC) for each gene and cell type. We find that evolutionary changes in gene expression in SF and ESF are highly correlated, supporting the hypothesis that the correlated gene expression changes are a prevalent feature of gene expression evolution. The experimental data set derives from a SCID mouse strain that was selected for slow cancer growth which led to substantial changes in the SF compared to wild type SCID mice. We isolated SF and ESF from wild type and evolved SCID mice and compared their gene expression profiles. We find a significant correlation between the gene expression contrasts of SF and ESF, which supports the hypothesis that gene expression variation in SF and ESF is correlated. We discuss the implications of these findings for the hypothesized correlation between placental invasiveness and vulnerability to metastatic cancer.

Extended lifespan in female Drosophila melanogaster through late-life caloric restriction.

Li, M. — 2023-07-17

Calorie restriction has many beneficial effects on healthspan and lifespan in a variety of species. However, how late in life application of caloric restriction can extend fly life is not clear. Here we show that late-life calorie restriction increases lifespan in female Drosophila melanogaster aged on a high calorie diet. This shift results in rapid decrease in mortality rate and extends fly lifespan. In contrast, shifting female flies from a low to a high calorie diet leads to a rapid increase in mortality and shorter lifespan. These changes are mediated by immediate metabolic and physiological adaptations. One of such adaptation is rapid adjustment in egg production, with flies directing excess energy towards egg production when shifted to a high diet, or away from reproduction in females shifted to low caloric diet. However, lifelong female fecundity reveals no associated fitness cost due to CR when flies are shifted to a high calorie diet. In view of high conservation of the beneficial effects of CR on physiology and lifespan in a wide variety of organisms, including humans, our findings could provide valuable insight into CR applications that could provide health benefits later in life.

CBD and PSP cell-passaged Tau Seeds Generate Heterogeneous Fibrils with A Subpopulation Adopting Disease Folds

Zeng, Z. — 2023-07-21

The recent discovery by cryo-electron microscopy (cryo-EM) that the neuropathological hallmarks of different tauopathies, including Alzheimers disease, corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP), are caused by unique misfolded conformations of the protein tau is among the most profound developments in neurodegenerative disease research. To capitalize on these discoveries for therapeutic development, one must achieve in vitro replication of tau fibrils that adopt the representative tauopathy disease folds, which represents a grand challenge for the field. An widely used approach is seeded propagation using pathological tau fibrils derived from post-mortem patients in biosensor cells that expresses a fragment of the tau protein, known as K18 or Tau4RD, containing mainly the microtubule-binding repeat domain of tau as the substrate. The new insight from cryo-EM raised the question whether the Tau4RD fragment is capable of adopting the specific tau folds found in CBD and PSP patient fibrils, and whether cell-passaged and amplified tau fibrils can be used as seeds to achieve cell-free assembly of recombinant 4R tau into fibrils without the addition of any cofactors. Using Double Electron Electron Resonance (DEER) spectroscopy, we discovered that cell-passaged pathological seeds generate heterogeneous fibrils that are distinct between the CBD and PSP lysate-seeded fibrils. These fibrils are also distinct from heparin-induced tau fibril populations. Moreover, the lysate-seeded fibrils contain a characteristic sub-population that resembles the disease fold corresponding to the respective starting patient sample. These findings indicate that templated propagation using CBD and PSP patient-derived fibrils is possible with a tau fragment that does not contain the entire pathological fibril core, and that additional mechanisms must be tuned to converge to a homogeneous fibril population.

Macrophage Xanthine Oxidoreductase Links LPS Induced Lung Inflammatory Injury to NLRP3 Inflammasome Expression and Mitochondrial Respiration

Fini, M. A. — 2023-07-21

Acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) remain poorly treated inflammatory lung disorders. Both reactive oxygen species (ROS) and macrophages are involved in the pathogenesis of ALI/ARDS. Xanthine oxidoreductase (XOR) is an ROS generator that plays a central role in the inflammation that contributes to ALI. To elucidate the role of macrophage-specific XOR in endotoxin induced ALI, we developed a conditional myeloid specific XOR knockout in mice. Myeloid specific ablation of XOR in LPS insufflated mice markedly attenuated lung injury demonstrating the essential role of XOR in this response. Macrophages from myeloid specific XOR knockout exhibited loss of inflammatory activation and increased expression of anti-inflammatory genes/proteins. Transcriptional profiling of whole lung tissue of LPS insufflated XORfl/fl//LysM-Cre mice demonstrated an important role for XOR in expression and activation of the NLRP3 inflammasome and acquisition of a glycolytic phenotype by inflammatory macrophages. These results identify XOR as an unexpected link between macrophage redox status, mitochondrial respiration and inflammatory activation.

Specific configurations of electrical synapses filter sensory information to drive choices in behavior

Almoril-Porras, A. — 2023-08-03

Synaptic configurations in precisely wired circuits underpin how sensory information is processed by the nervous system, and the emerging animal behavior. This is best understood for chemical synapses, but far less is known about how electrical synaptic configurations modulate, in vivo and in specific neurons, sensory information processing and context-specific behaviors. We discovered that INX-1, a gap junction protein that forms electrical synapses, is required to deploy context-specific behavioral strategies during C. elegans thermotaxis behavior. INX-1 couples two bilaterally symmetric interneurons, and this configuration is required for the integration of sensory information during migration of animals across temperature gradients. In inx-1 mutants, uncoupled interneurons display increased excitability and responses to subthreshold temperature stimuli, resulting in abnormally longer run durations and context-irrelevant tracking of isotherms. Our study uncovers a conserved configuration of electrical synapses that, by increasing neuronal capacitance, enables differential processing of sensory information and the deployment of context-specific behavioral strategies. One-Sentence SummaryCoupling of interneurons by electrical synapses reduces membrane resistance and filters sensory inputs to guide sensory-dependent behavioral choices.

A pro-inflammatory stem cell niche drives myelofibrosis through a targetable galectin 1 axis

Li, R. — 2023-08-07

Myeloproliferative neoplasms are stem cell-driven cancers associated with a large burden of morbidity and mortality. The majority of patients present with early-stage disease, but a substantial proportion progress to myelofibrosis and/or secondary leukemia, advanced cancers with a poor prognosis and high symptom burden. Currently, it remains difficult to predict progression, and we lack therapies that reliably prevent or reverse fibrosis development. A major bottleneck to the discovery of disease-modifying therapies has been an incomplete understanding of the interplay between perturbed cellular and molecular states. Several cell types have individually been implicated, but a comprehensive analysis of myelofibrotic bone marrow is lacking. We therefore mapped the crosstalk between bone marrow cell types in myelofibrotic bone marrow. We found that inflammation and fibrosis are orchestrated by a quartet of immune and stromal cell lineages - with basophils and mast cells creating a TNF signaling hub, communicating with megakaryocytes, mesenchymal stromal cells and pro-inflammatory fibroblasts. We identified the y-galactoside binding protein galectin 1 as a striking biomarker of progression to myelofibrosis and poor survival in multiple patient cohorts, and as a promising therapeutic target, with reduced myeloproliferation and fibrosis in vitro and in vivo and improved survival following galectin 1 inhibition. In human bone marrow organoids, TNF increased galectin 1 expression, suggesting a feedback loop wherein the pro-inflammatory MPN clone creates a self-reinforcing niche, fueling progression to advanced disease. This study provides a valuable resource for studying hematopoietic cell-niche interactions, with broad relevance for cancer-associated inflammation and disorders of tissue fibrosis.

Single nucleotide polymorphisms and Zn transport by nuclear ZIP11 shape cancer phenotypes in HeLa cells

Kim, E. Y. — 2023-08-15

Zinc (Zn) is an essential micronutrient that regulates critical biological processes such as enzymatic function, gene expression, and cell signaling and provides structural stability to proteins. Under physiological conditions, Zn is a divalent cation (Zn2+) in an inactive redox state. Maintaining Zn homeostasis is essential for normal cell development and function, and any dysregulation in supply and transport can lead to pathophysiological conditions. Zn transporters, such as ZIP11, are critical regulators in Zn homeostasis. In mammals, ZIP11 belongs to the GufA subfamily of ZIP transporters and is primarily found in the nucleus and the Golgi apparatus. Our laboratory recently reported an essential role of ZIP11 in maintaining nuclear Zn levels in the cervical cancer cell line HeLa that supports various hallmark phenotypes of cancer. Genomic analysis of publicly available cervical and ovarian cancer patient datasets identified several single-nucleotide polymorphisms (SNPs) in the ZIP11 coding region that correlated with disease severity. We hypothesized that these SNPs might have potentially deleterious consequences as they are in coding regions that could affect ZIP11 function by increasing substrate accessibility, potentially enhancing the carcinogenic phenotype of HeLa cells. In addition, we identified a classic Zn-metal binding site (MBS) composed of three relevant residues which may be required for transmembrane Zn-transport, maintenance of metal homeostasis, and the carcinogenic properties of HeLa cells. To address these questions, we utilized our well-established model of stably knock down (KD) ZIP11 in HeLa cells and overexpressed ZIP11 encoding single mutations corresponding to the SNPs and the MBS. Overexpression of ZIP11 encoding SNPs restored the Zn levels and the proliferation, migration, and invasive defects of ZIP11 KD cells. On the other hand, cells expressing ZIP11 with single MBS mutations exhibited a phenotype similar to KD cells, suggesting that Zn transport by this transporter is necessary for establishing and maintaining carcinogenic properties. The work highlights the functional relevance of nuclear Zn transport by ZIP11 to maintain homeostasis and support carcinogenic properties in ovarian cancer cells.

Generation of isogenic models of Angelman syndrome and Prader-Willi syndrome in CRISPR/Cas9-engineered human embryonic stem cells

Gilmore, R. B. — 2023-08-30

Angelman Syndrome (AS) and Prader-Willi Syndrome (PWS), two distinct neurodevelopmental disorders, result from loss of expression from imprinted genes in the chromosome 15q11-13 locus most commonly caused by a megabase-scale deletion on either the maternal or paternal allele, respectively. Each occurs at an approximate incidence of 1/15,000 to 1/30,000 live births and has a range of debilitating phenotypes. Patient-derived induced pluripotent stem cells (iPSCs) have been valuable tools to understand human-relevant gene regulation at this locus and have contributed to the development of therapeutic approaches for AS. Nonetheless, gaps remain in our understanding of how these deletions contribute to dysregulation and phenotypes of AS and PWS. Variability across cell lines due to donor differences, reprogramming methods, and genetic background make it challenging to fill these gaps in knowledge without substantially increasing the number of cell lines used in the analyses. Isogenic cell lines that differ only by the genetic mutation causing the disease can ease this burden without requiring such a large number of cell lines. Here, we describe the development of isogenic human embryonic stem cell (hESC) lines modeling the most common genetic subtypes of AS and PWS. These lines allow for a facile interrogation of allele-specific gene regulation at the chromosome 15q11-q13 locus. Additionally, these lines are an important resource to identify and test targeted therapeutic approaches for patients with AS and PWS.

Unraveling human hematopoietic progenitor cell diversity through association with intrinsic regulatory factors

Favaro, P. — 2023-08-31

Hematopoietic stem and progenitor cell (HSPC) transplantation is an essential therapy for hematological conditions, but finer definitions of human HSPC subsets with associated function could enable better tuning of grafts and more routine, lower-risk application. To deeply phenotype HSPCs, following a screen of 328 antigens, we quantified 41 surface proteins and functional regulators on millions of CD34+ and CD34- cells, spanning four primary human hematopoietic tissues: bone marrow, mobilized peripheral blood, cord blood, and fetal liver. We propose more granular definitions of HSPC subsets and provide new, detailed differentiation trajectories of erythroid and myeloid lineages. These aspects of our revised human hematopoietic model were validated with corresponding epigenetic analysis and in vitro clonal differentiation assays. Overall, we demonstrate the utility of using molecular regulators as surrogates for cellular identity and functional potential, providing a framework for description, prospective isolation, and cross-tissue comparison of HSPCs in humans.

Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19

Padi, S. K. — 2023-08-31

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation.1 Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases,2 while mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55.3 While the role of kinases in mitotic entry is well-established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited.4 For PP2A:B55, inhibition is achieved by the two intrinsically disordered proteins (IDPs), ARPP19 (phosphorylation-dependent)6,7 and FAM122A5 (inhibition is phosphorylation-independent). Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies both IDPs bind PP2A:B55, but do so in highly distinct manners, unexpectedly leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provides a molecular roadmap for the development of therapeutic interventions for PP2A:B55 related diseases.

The Implant-Induced Foreign Body Response is Limited by CD13-Dependent Regulation of Ubiquitination of Fusogenic Proteins

Ghosh, M. — 2023-09-21

Implanted medical devices from artificial heart valves, arthroscopic joints to implantable sensors often induce a Foreign Body Response (FBR), a form of chronic inflammation resulting from the inflammatory reaction to a persistent foreign stimulus. The FBR is characterized by a subset of multinucleated giant cells (MGCs) formed by macrophage fusion, the Foreign Body Giant cells (FBGCs), accompanied by inflammatory cytokines, matrix deposition and eventually, deleterious fibrotic implant encapsulation. Despite efforts to improve biocompatibility, implant-induced FBR persists, compromising the utility of devices and making efforts to control the FBR imperative for long-term function. Controlling macrophage fusion in FBGC formation presents a logical target to prevent implant failure, but the actual contribution of FBGCs to FBR-induced damage is controversial. CD13 is a molecular scaffold and in vitro induction of CD13KO bone-marrow progenitors generates many more MGCs than WT, suggesting CD13 regulates macrophage fusion. Moreover, in the mesh implant model of FBR, CD13KO mice produced significantly more peri-implant FBGCs with enhanced TGF{beta} expression and increased collagen deposition vs. WT. Pre-fusion, increased protrusion and microprojection formation accompanies hyperfusion in the absence of CD13. Expression of fusogenic proteins driving cell-cell fusion was aberrantly sustained at high levels in CD13KO MGCs, which we show is due to a novel CD13 function, regulating ubiquitin/proteasomal protein degradation. By controlling protein degradation, CD13 becomes a physiologic brake preventing aberrant macrophage fusion and may be a novel therapeutic target to improve success of implanted medical devices. Furthermore, our data directly implicates FBGCs in the detrimental fibrosis that characterizes the FBR.

Identifying key underlying regulatory networks and predicting targets of orphan C/D box SNORD116 snoRNAs in Prader-Willi syndrome

Gilmore, R. B. — 2023-10-05

Prader-Willi syndrome (PWS) is a rare neurodevelopmental disorder characterized principally by initial symptoms of neonatal hypotonia and failure-to-thrive in infancy, followed by hyperphagia and obesity. It is well established that PWS is caused by loss of paternal expression of the imprinted region on chromosome 15q11-q13. While most PWS cases exhibit megabase-scale deletions of the paternal chromosome 15q11-q13 allele, several PWS patients have been identified harboring a much smaller deletion encompassing primarily SNORD116. This finding suggests SNORD116 is a direct driver of PWS phenotypes. The SNORD116 gene cluster is composed of 30 copies of individual SNORD116 C/D box small nucleolar RNAs (snoRNAs). Many C/D box snoRNAs have been shown to guide chemical modifications of other RNA molecules, often ribosomal RNA (rRNA). However, SNORD116 snoRNAs are termed orphans because no verified targets have been identified and their sequences show no significant complementarity to rRNA. It is crucial to identify the targets and functions of SNORD116 snoRNAs because all reported PWS cases lack their expression. To address this, we engineered two different deletions modelling PWS in two distinct human embryonic stem cell (hESC) lines to control for effects of genetic background. Utilizing an inducible expression system enabled quick, reproducible differentiation of these lines into neurons. Systematic comparisons of neuronal gene expression across deletion types and genetic backgrounds revealed a novel list of 42 consistently dysregulated genes. Employing the recently described computational tool snoGloBe, we discovered these dysregulated genes are significantly enriched for predicted SNORD116 targeting versus multiple control analyses. Importantly, our results showed it is critical to use multiple isogenic cell line pairs, as this eliminated many spuriously differentially expressed genes. Our results indicate a novel gene regulatory network controlled by SNORD116 is likely perturbed in PWS patients.

ROCK1 activates mitochondrial fission leading to oxidative stress and muscle atrophy

Si, M. — 2023-10-22

Chronic kidney disease (CKD) is often associated with protein-energy wasting (PEW), which is characterized by a reduction in muscle mass and strength. Although mitochondrial dysfunction and oxidative stress have been implicated to play a role in the pathogenesis of muscle wasting, the underlying mechanisms remain unclear. In this study, we used transcriptomics, metabolomics analyses and mouse gene manipulating approaches to investigate the effects of mitochondrial plasticity and oxidative stress on muscle wasting in mouse CKD models. Our results showed that the expression of oxidative stress response genes was increased, and that of oxidative phosphorylation genes was decreased in the muscles of mice with CKD. This was accompanied by reduced oxygen consumption rates, decreased levels of mitochondrial electron transport chain proteins, and increased cellular oxidative damage. Excessive mitochondrial fission was also observed, and we found that the activation of ROCK1 was responsible for this process. Inducible expression of muscle-specific constitutively active ROCK1 (mROCK1ca) exacerbated mitochondrial fragmentation and muscle wasting in CKD mice. Conversely, ROCK1 depletion (ROCK1-/-) alleviated these phenomena. Mechanistically, ROCK1 activation promoted the recruitment of Drp1 to mitochondria, thereby facilitating fragmentation. Notably, the pharmacological inhibition of ROCK1 mitigated muscle wasting by suppressing mitochondrial fission and oxidative stress. Our findings demonstrate that ROCK1 participates in CKD-induced muscle wasting by promoting mitochondrial fission and oxidative stress, and pharmacological suppression of ROCK1 could be a therapeutic strategy for combating muscle wasting in CKD conditions. Translational StatementProtein-energy wasting (PEW) is a prevalent issue among patients with chronic kidney disease (CKD) and is characterized by the loss of muscle mass. Our research uncovers a critical role that ROCK1 activation plays in muscle wasting induced by CKD. We found that ROCK1 is instrumental in causing mitochondrial fission, which leads to increased oxidative stress in muscle cells. By employing a pharmacological inhibitor, hydroxyfasudil, we were able to effectively curb ROCK1 activity, which in turn mitigated muscle wasting by reducing both mitochondrial fission and oxidative stress. These findings suggest that pharmacological inhibition of ROCK1 presents a promising therapeutic strategy for combating the muscle wasting associated with CKD.

Transcriptional Control of Neocortical Size and Microcephaly

Barao, S. — 2023-11-02

The mammalian neocortex differs vastly in size and complexity between mammalian species, yet the mechanisms that lead to an increase in brain size during evolution are not known. We show here that two transcription factors coordinate gene expression programs in progenitor cells of the neocortex to regulate their proliferative capacity and neuronal output in order to determine brain size. Comparative studies in mice, ferrets and macaques demonstrate an evolutionary conserved function for these transcription factors to regulate progenitor behaviors across the mammalian clade. Strikingly, the two transcriptional regulators control the expression of large numbers of genes linked to microcephaly suggesting that transcriptional deregulation as an important determinant of the molecular pathogenesis of microcephaly, which is consistent with the finding that genetic manipulation of the two transcription factors leads to severe microcephaly.

Immunosuppressive tumor microenvironment of osteosarcoma

Taylor, A. M. — 2023-11-03

Osteosarcoma is the most common malignant bone tumor in children, characterized by a high degree of genomic instability, resulting in copy-number alterations and genomic rearrangements without disease-defining recurrent mutations. Clinical trials based on molecular characterization have failed to find new effective therapies or improve outcomes over the last 40 years. To better understand the immune microenvironment of osteosarcoma, we performed single-cell RNA sequencing on six tumor biopsy samples, combined with a previously-published cohort of six samples. Additional osteosarcoma samples were profiled using spatial transcriptomics for validation of discovered subtypes and to add spatial context. Analysis revealed immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs), regulatory and exhausted T-cells, and LAMP3+ dendritic cells. Using cell-cell communication modeling, we identified robust interactions between MDSCs and other cells, leading to NF-{kappa}B upregulation and an immunosuppressive microenvironment, as well as interactions involving regulatory T-cells and osteosarcoma cells that promoted tumor progression and a proangiogenic niche. Statement of SignificanceOsteosarcoma patient survival has remained stagnant for several decades due to lack of successful therapy for high-risk patients, including those with metastatic disease. Identifying novel therapeutics including immunotherapies is of great clinical importance. Our study highlights several important immunosuppressive mechanisms within osteosarcoma that should be considered when developing future immunotherapies.

Molecular basis for dual functions in pilus assembly modulated by the lid of a pilus-specific sortase

Chang, C. — 2023-11-05

The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-[A] resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.

NaP-TRAP, a novel massively parallel reporter assay to quantify translation control.

Strayer, E. C. — 2023-11-09

The cis-regulatory elements encoded in a mRNA determine its stability and translational output. While there has been a considerable effort to understand the factors driving mRNA stability, the regulatory frameworks governing translational control remain elusive. We have developed a novel massively parallel reporter assay (MPRA) to measure mRNA translation, Nascent Peptide Translating Ribosome Affinity Purification (NaP-TRAP). NaP-TRAP measures translation in a frame specific manner through the immunocapture of epitope tagged nascent peptides of reporter mRNAs. In contrast to existing MPRA methods, NaP-TRAP does not require specialized equipment and is readily adaptable to steady-state and dynamic model systems. We have employed NaP-TRAP to quantify Kozak strength and the regulatory landscapes of 5 UTRs in the developing zebrafish embryo and in human cells, characterizing general and developmentally dynamic cis-regulatory elements. To this end, we identify U-rich motifs as general enhancers, and upstream ORFs and GC-rich motifs as global repressors of translation. We also observe a translational switch during the maternal-to-zygotic transition, where C-rich motifs shift from repressors to prominent activators of translation. Conversely, we show that microRNA sites in the 5 UTR repress translation following the zygotic expression of miR-430. Together these results demonstrate that NaP-TRAP is a versatile, accessible, and powerful method to decode the regulatory functions of UTRs across different systems.

SMAD4 promotes formation of terminally differentiated CTLs that localize in the microvasculature of the lungs but are excluded from the lumen of the airways

Suarez-Ramirez, J. — 2023-11-17

Cytotoxic T lymphocytes (CTLs) circulate around the body searching for infected and transformed cells, that undergo apoptosis when lytic granules are delivered into the cytoplasm. To find pathogens that propagate in different tissues, naive CD8 T cells differentiate into heterogeneous populations of effector (TEFF) and memory CD8 T cells with different migratory properties. Several subsets can be identified using antibodies that recognize surface receptors that are expressed at specific stages during CD8 T cell differentiation. Although flow cytometry is a powerful method for tracking antigen specific CTLs during a dynamic immune response, the data provide little information about the distribution of cells in specific anatomical compartments. In this study, confocal imaging was used to explore how signaling via SMAD4 influenced the tissue-tropism of antigen specific CTLs during respiratory infection. During microbial infection, wildtype CTLs gave rise to terminally differentiated TEFF cells that expressed KLRG1 and CX3CR1 at high levels and localized in the microvasculature of the lungs. However, both markers were expressed at reduced levels on SMAD4-deficient CTLs, which preferentially entered the lumen of the airways. These disparate homing properties emphasize the important contributions of SMAD signaling pathways to cell-mediated immunity.

Insight into the gut virome in patients with multiple sclerosis

Bokoliya, S. C. — 2023-11-20

Multiple sclerosis (MS) is an autoimmune condition associated with dysbiosis in the bacterial element of microbiome, yet limited information exists regarding dysbiosis in the virome. In this study, we examined the virome in 20 relapsing-remitting MS (RRMS) patients and 22 healthy controls (HC). We extracted virus-like particles (VLP) genomic DNA through sequential filtration, followed by deep metagenomic sequencing approaches with and without multiple displacement amplification (MDA). We found significantly lower diversity in the gut virome of RRMS patients relative to HC, consistent across both sequencing methods. MDA method identified reduced relative abundance of Microviridae and Myoviridae bacteriophage, and eukaryotic virus such as Herpesviridae and Phycodnaviridae in RRMS patients compared to HC. Non-MDA methods showed reduction in relative abundance of Siphoviridae bacteriophage and eukaryotic viruses such as Ackermannviridae, Demerecviridae, Dicistroviridae, Herelleviridae, Mesnidovirineae in RRMS patients. Cluster analysis revealed that the whole virome family was dominated by Podoviridae and Siphoviridae clusters. Comparing dietary metadata between these clusters, RRMS patients in the Siphoviridae-dominated Cluster B showed significantly higher consumption of refined grains and salad dressings compared to those in the Podoviridae-dominated Cluster A. Correlation analysis between gut viruses and bacteria demonstrated that Siphoviridae exhibited positive correlations with many different bacterial genera. Conversely, Microviridae displayed negative correlations with many different bacterial genera. These findings underscore the alterations in viral diversity and taxonomic composition of the gut virome in RRMS patients. Our study represents the first step in understanding the gut virome in MS patients, providing a groundwork for future research on the role of the gut virome in the context of MS.

Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes

Scardigli, M. — 2023-11-30

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-coordinated excitation-contraction coupling (ECC) and, thus, homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarisation is active (local generation of action potentials; AP) or passive (following depolarisation of the outer cell surface sarcolemma; SS) has not been experimentally assessed in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesise that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were electrically insulated from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTSO_LICardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, ensuring synchronous and uniform cell contraction. C_LIO_LIIn this study, we investigate whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. C_LIO_LIFor this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of sub-cellular membrane domains. C_LIO_LIOur findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. C_LIO_LIThis active electrical capability may serve as a protective mechanism against voltage drops occurring within the T-tubular network. C_LI

Oligodendrocytes and neurons contribute to amyloid-β deposition in Alzheimer's disease

Sasmita, A. O. — 2023-12-11

In Alzheimers disease (AD), amyloid-{beta} (A{beta}) is thought to be of neuronal origin. However, in single-cell RNAseq datasets from mouse and human, we found transcripts of amyloid precursor protein (APP) and the amyloidogenic-processing machinery equally abundant in oligodendrocytes (OLs). By cell-type-specific deletion of Bace1 in a humanized knock-in AD model, APPNLGF, we demonstrate that almost a third of cortical A{beta} deposited in plaques is derived from OLs. However, excitatory projection neurons must provide a threshold level of A{beta} production for plaque deposition to occur and for oligodendroglial A{beta} to co-aggregate. Indeed, very few plaques are deposited in the absence of neuronally-derived A{beta}, although soluble A{beta} species are readily detected, especially in subcortical white matter. Our data identify OLs as a source of A{beta} in vivo and further underscore a non-linear relationship between cellular A{beta} production and resulting plaque formation. Ultimately, our observations are relevant for therapeutic strategies aimed at disease prevention in AD.

IMPDH2 filaments protect from neurodegeneration in AMPD2 deficiency

Flores-Mendez, M. — 2024-01-22

Metabolic dysregulation is one of the most common causes of pediatric neurodegenerative disorders. However, how the disruption of ubiquitous and essential metabolic pathways predominantly affect neural tissue remains unclear. Here we use mouse models of AMPD2 deficiency to study cellular and molecular mechanisms that lead to selective neuronal vulnerability to purine metabolism imbalance. We show that AMPD deficiency in mice primarily leads to hippocampal dentate gyrus degeneration despite causing a generalized reduction of brain GTP levels. Remarkably, we found that neurodegeneration resistant regions accumulate micron sized filaments of IMPDH2, the rate limiting enzyme in GTP synthesis. In contrast, IMPDH2 filaments are barely detectable in the hippocampal dentate gyrus, which shows a progressive neuroinflammation and neurodegeneration. Furthermore, using a human AMPD2 deficient neural cell culture model, we show that blocking IMPDH2 polymerization with a dominant negative IMPDH2 variant, impairs AMPD2 deficient neural progenitor growth. Together, our findings suggest that IMPDH2 polymerization prevents detrimental GTP deprivation in neurons with available GTP precursor molecules, providing resistance to neurodegeneration. Our findings open the possibility of exploring the involvement of IMPDH2 assembly as a therapeutic intervention for neurodegeneration.

Oscillatory Hypoxia Induced Unfolded Protein Folding Response Gene Expression Predicts Low Survival in Human Breast Cancer Patients

Suhail, Y. — 2024-01-26

Hypoxia is one of the key factors in the tumor microenvironment regulating nearly all steps in the metastatic cascade in many cancers, including in breast cancer. The hypoxic regions can however be dynamic with the availability of oxygen fluctuating or oscillating. The canonical response to hypoxia is relayed by transcription factor HIF-1, which is stabilized in hypoxia and acts as the master regulator of a large number of downstream genes. However, HIF-1 transcriptional activity can also fluctuate either due to unstable hypoxia, or by lactate mediated non-canonical degradation of HIF-1. Our understanding of how oscillatory hypoxia or HIF-1 activity specifically influence cancer malignancy is very limited. Here, using MDA-MB-231 cells as a model of triple negative breast cancer characterized by severe hypoxia, we measured the gene expression changes induced specifically by oscillatory hypoxia. We found that oscillatory hypoxia can specifically regulate gene expression differently, and at times opposite to stable hypoxia. Using The Cancer Genome Atlas (TCGA) RNAseq data of human cancer samples, we show that the oscillatory specific gene expression signature in MDA-MB-231 is enriched in most human cancers, and prognosticate low survival in breast cancer patients. In particular, we found that oscillatory hypoxia, unlike stable hypoxia, induces unfolded protein folding response (UPR) in cells resulting in gene expression predicting reduced survival.

Computation-aided Design of Rod-Shaped Janus Base Nanopieces for Improved Tissue Penetration and Therapeutics Delivery

Lee, J. — 2024-01-27

Despite the development of various drug delivery technologies, there remains a significant need for vehicles that can improve targeting and biodistribution in "hard-to-penetrate" tissues. Some solid tumors, for example, are particularly challenging to penetrate due to their dense extracellular matrix (ECM). In this study, we have formulated a new family of rod-shaped delivery vehicles named Janus base nanopieces (Rod JBNps), which are more slender than conventional spherical nanoparticles, such as lipid nanoparticles (LNPs). These JBNp nanorods are formed by bundles of DNA-inspired Janus base nanotubes (JBNts) with intercalated delivery cargoes. To develop this novel family of delivery vehicles, we employed a computation-aided design (CAD) methodology that includes molecular dynamics and response surface methodology. This approach precisely and efficiently guides experimental designs. Using an ovarian cancer model, we demonstrated that JBNps markedly improve penetration into the dense ECM of solid tumors, leading to better treatment outcomes compared to FDA-approved spherical LNP delivery. This study not only successfully developed a rod-shaped delivery vehicle for improved tissue penetration but also established a CAD methodology to effectively guide material design.

vSPACE: Exploring Virtual Spatial Representation of Articular Chondrocytes at the Single-Cell Level

Zhang, C. — 2024-02-10

Single cell RNA sequencing technology has been dramatically changing how gene expression studies are performed. However, its use has been limited to identifying subtypes of cells by comparing cells gene expression levels in an unbiased manner to produce a 2D plot (e.g., UMAP/tSNE). We developed a new method of placing cells in 2D space. This system, called vSPACE, shows a virtual spatial representation of scRNAseq data obtained from human articular cartilage by emulating the concept of spatial transcriptomics technology, but virtually. This virtual 2D plot presentation of human articular cartage cells generates several zonal distribution patterns, in one or multiple genes at a time, reveling patterns that scientists can appreciate as imputed spatial distribution patterns along the zonal axis. The discovered patterns are explainable and remarkably consistent across all six healthy doners despite their respectively different clinical variables (age and sex), suggesting the confidence of the discovered patterns.

Stable and Oscillatory Hypoxia Differentially Regulate Invasibility of Breast Cancer Associated Fibroblasts

Du, W. — 2024-03-28

As local regions in the tumor outstrip their oxygen supply, hypoxia can develop, affecting not only the cancer cells, but also other cells in the microenvironment, including cancer associated fibroblasts (CAFs). Hypoxia is also not necessarily stable over time, and can fluctuate or oscillate. Hypoxia Inducible Factor-1 is the master regulator of cellular response to hypoxia, and can also exhibit oscillations in its activity. To understand how stable, and fluctuating hypoxia influence breast CAFs, we measured changes in gene expression in CAFs in normoxia, hypoxia, and oscillatory hypoxia, as well as measured change in their capacity to resist, or assist breast cancer invasion. We show that hypoxia has a profound effect on breast CAFs causing activation of key pathways associated with fibroblast activation, but reduce myofibroblast activation and traction force generation. We also found that oscillatory hypoxia, while expectedly resulted in a "sub-hypoxic" response in gene expression, it resulted in specific activation of pathways associated with actin polymerization and actomyosin maturation. Using traction force microscopy, and a nanopatterned stromal invasion assay, we show that oscillatory hypoxia increases contractile force generation vs stable hypoxia, and increases heterogeneity in force generation response, while also additively enhancing invasibility of CAFs to MDA-MB-231 invasion. Our data show that stable and unstable hypoxia can regulate many mechnobiological characteristics of CAFs, and can contribute to transformation of CAFs to assist cancer dissemination and onset of metastasis.

Profiling microRNA expression during senescence and aging: mining for a diagnostic tool of senescent-cell burden

Weigl, M. — 2024-04-10

In the last decade cellular senescence, a hallmark of aging, has come into focus for pharmacologically targeting aging processes. Senolytics are one of these interventive strategies that have advanced into clinical trials, creating an unmet need for minimally invasive biomarkers of senescent cell load to identify patients at need for senotherapy. We created a landscape of miRNA and mRNA expression in five human cell types induced to senescence in-vitro and provide proof-of-principle evidence that miRNA expression can track senescence burden dynamically in-vivo using transgenic p21high senescent cell clearance in HFD fed mice. Finally, we profiled miRNA expression in seven different tissues, total plasma, and plasma derived EVs of young and 25 months old mice. In a systematic analysis, we identified 22 candidate senomiRs with potential to serve as circulating biomarkers of senescence not only in rodents, but also in upcoming human clinical senolytic trials.

Asymmetric stem cell division maintains the genetic heterogeneity of tissue cells

Bener, M. B. — 2024-05-16

Within a given tissue, the stem cell niche provides the microenvironment for stem cells suitable for their self-renewal. Conceptually, the niche space constrains the size of a stem-cell pool, as the cells sharing the niche compete for its space. It has been suggested that either neutral-or non-neutral-competition of stem cells changes the clone dynamics of stem cells. Theoretically, if the rate of asymmetric division is high, the stem cell competition is limited, thus suppressing clonal expansion. However, the effects of asymmetric division on clone dynamics have never been experimentally tested. Here, using the Drosophila germline stem cell (GSC) system, as a simple model of the in-vivo niche, we examine the effect of division modes (asymmetric or symmetric) on clonal dynamics by combining experimental approaches with mathematical modeling. Our results suggest that the rate of asymmetric division positively correlates with the time a stem cell clone takes to expand. Taken together, our data suggests that asymmetric division is essential for maintaining the genetic variation of stem cells and thus serves as a critical mechanism for safeguarding fertility over the animal age or preventing multiple disorders caused by the clonal expansion of stem cells.

mosGraphGen: a novel tool to generate multi-omic signaling graphs to facilitate integrative and interpretable graph AI model development

Zhang, H. — 2024-05-18

Multi-omics data, i.e., genomics, epigenomics, transcriptomics, proteomics, characterize cellular complex signaling systems from multi-level and multi-view and provide a holistic view of complex cellular signaling pathways. However, it remains challenging to integrate and interpret multi-omics data for mining key disease targets and signaling pathways. Graph AI models have been widely used to analyze graph-structure datasets, and are ideal for integrative multi-omics data analysis because they can naturally integrate and represent multi-omics data as a biologically meaningful multi-level signaling graph and interpret multi-omics data via graph node and edge ranking analysis. However, it is non-trivial for graph-AI model developers to pre-analyze multi-omics data and convert the data into biologically meaningful graphs, which can be directly fed into graph-AI models. To resolve this challenge, we developed mosGraphGen (multi-omics signaling graph generator), generating Multi-omics Signaling graphs (mos-graph) of individual samples by mapping multi-omics data onto a biologically meaningful multi-level background signaling network with data normalization by aggregating measurements and aligning to the reference genome. With mosGraphGen, AI model developers can directly apply and evaluate their models using these mos-graphs. In the results, mosGraphGen was used and illustrated using two widely used multi-omics datasets of TCGA and Alzheimers disease (AD) samples. The code of mosGraphGen is open-source and publicly available via GitHub: https://github.com/FuhaiLiAiLab/mosGraphGen

Role of the nucleus accumbens in signaled avoidance actions

Zhou, J. — 2024-06-01

Animals, humans included, navigate their environments guided by sensory cues, responding adaptively to potential dangers and rewards. Avoidance behaviors serve as adaptive strategies in the face of signaled threats, but the neural mechanisms orchestrating these behaviors remain elusive. Current circuit models of avoidance behaviors indicate that the nucleus accumbens (NAc) in the ventral striatum plays a key role in signaled avoidance behaviors, but the nature of this engagement is unclear. Evolving perspectives propose the NAc as a pivotal hub for action selection, integrating cognitive and affective information to heighten the efficiency of both appetitive and aversive motivated behaviors. To unravel the engagement of the NAc during active and passive avoidance, we used calcium imaging fiber photometry and single-unit recordings to examine NAc GABAergic neuron activity in freely moving mice performing avoidance behaviors. We then probed the functional significance of NAc neurons using optogenetics, and genetically targeted or electrolytic lesions. We found that NAc neurons code contraversive orienting movements and avoidance actions. Intriguingly, specific optogenetic patterns intended to excite NAc GABAergic neurons resulted in local somatic inhibition through GABAergic synaptic collaterals. Nevertheless, these patterns directly excited NAc GABAergic output axons, which in turn inhibited their targets, disrupting active avoidance behavior. Thus, this disruption stemmed from abnormal alterations in the activity of downstream midbrain areas crucial for the behavior. In contrast, direct optogenetic inhibition or lesions of NAc neurons did not impair active or passive avoidance behaviors, challenging the notion of their purported pivotal role in adaptive avoidance. The findings emphasize that NAc is not required for avoidance behaviors, but disruptions in NAc output during pathological states can impair these behaviors.

Chioso: Segmentation-free Annotation of Spatial Transcriptomics Data at Sub-cellular Resolution via Adversarial Learning

Yu, J. — 2024-06-04

Recent advances in spatial transcriptomics technology have produced full-transcriptomic scale dataset with subcellular spatial resolutions. Here we present a new computational algorithm, chioso, that can transfer cell-level labels from a reference dataset (typically a single-cell RNA sequencing dataset) to a target spatial dataset by assigning a label to every spatial location at sub-cellular resolution. Importantly, we do this without requiring single cell segmentation inputs, thereby simplifying the experiments, and allowing for a more streamlined, and potentially more accurate, analysis pipeline. Using a generative neural network as the underlying algorithmic engine, chioso is very fast and scales well to large datasets. We validated the performance of chioso using synthetic data and further demonstrated its scalability by analyzing the complete MOSTA dataset acquired using the Stereo-Seq technology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/597195v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1521a38org.highwire.dtl.DTLVardef@1376191org.highwire.dtl.DTLVardef@e6b4c3org.highwire.dtl.DTLVardef@65801b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Modeling differences in neurodevelopmental maturity of the reading network using support vector regression on functional connectivity data

Lasnick, O. H. M. — 2024-06-11

The construction of growth charts trained to predict age or developmental deviation (the brain-age index) based on structural/functional properties of the brain may be informative of childrens neurodevelopmental trajectories. When applied to both typically and atypically developing populations, results may indicate that a particular condition is associated with atypical maturation of certain brain networks. Here, we focus on the relationship between reading disorder (RD) and maturation of functional connectivity (FC) patterns in the prototypical reading/language network using a cross-sectional sample of N = 742 participants aged 6-21 years. A support vector regression model is trained to predict chronological age from FC data derived from a whole-brain model as well as multiple reduced models, which are trained on FC data generated from a successively smaller number of regions in the brains reading network. We hypothesized that the trained models would show systematic underestimation of brain network maturity for poor readers, particularly for the models trained with reading/language regions. Comparisons of the different models predictions revealed that while the whole-brain model outperforms the others in terms of overall prediction accuracy, all models successfully predicted brain maturity, including the one trained with the smallest amount of FC data. In addition, all models showed that reading ability affected the brain-age gap, with poor readers ages being underestimated and advanced readers ages being overestimated. Exploratory results demonstrated that the most important regions and connections for prediction were derived from the default mode and frontoparietal control networks. GlossaryDevelopmental dyslexia / reading disorder (RD): A specific learning disorder affecting reading ability in the absence of any other explanatory condition such as intellectual disability or visual impairment Support vector regression (SVR): A supervised machine learning technique which predicts continuous outcomes (such as chronological age) rather than classifying each observation; finds the best-fit function within a defined error margin Principal component analysis (PCA): A dimensionality reduction technique that transforms a high-dimensional dataset with many features per observation into a reduced set of principal components for each observation; each component is a linear combination of several original (correlated) features, and the final set of components are all orthogonal (uncorrelated) to one another Brain-age index: A numerical index quantifying deviation from the brains typical developmental trajectory for a single individual; may be based on a variety of morphometric or functional properties of the brain, resulting in different estimates for the same participant depending on the imaging modality used Brain-age gap (BAG): The difference, given in units of time, between a participants true chronological age and a predictive models estimated age for that participant based on brain data (Actual - Predicted); may be used as a brain-age index HighlightsO_LIA machine learning model trained on functional data predicted participants ages C_LIO_LIThe model showed variability in age prediction accuracy based on reading skills C_LIO_LIThe model highly weighted data from frontoparietal and default mode regions C_LIO_LINeural markers of reading and language are diffusely represented in the brain C_LI

Multi-parametric thrombus profiling microfluidics detects intensified biomechanical thrombogenesis associated with hypertension and aging

Din, M. — 2024-06-12

Arterial thrombosis, which represents a critical complication of cardiovascular diseases, is a leading cause of death and disability worldwide with no effective bioassay for clinical prediction. As a symbolic feature of arterial thrombosis, severe stenosis in the blood vessel creates a high-shear, high-gradient flow environment that effectively facilitates platelet aggregation towards vessel occlusion even with platelet amplification loops inhibited. However, no approach is currently available to comprehensively characterize the size, composition and platelet activation status of thrombi forming under this biorheological condition. Here, we present a thrombus profiling assay that monitors the multi-dimensional attributes of thrombi forming in conditions mimicking the physiological scenario of arterial thrombosis. Using this platform, we demonstrate that different receptor-ligand interactions contribute distinctively to the composition and activation status of the thrombus. Our investigation into hypertensive and older individuals reveals intensified biomechanical thrombogenesis and multi-dimensional thrombus profile abnormalities, demonstrating a direct contribution of mechanobiology to arterial thrombosis and endorsing the diagnostic potential of the assay. Furthermore, we identify the hyperactivity of GPIb-integrin IIb{beta}3 mechanosensing axis as a molecular mechanism that contributes to hypertension-associated arterial thrombosis. By studying the interactions between anti-thrombotic inhibitors and hypertension, and the inter-individual variability in personal thrombus profiles, our work reveals a critical need for personalized anti-thrombotic drug selection that accommodates each patients pathological profile.

Inferring single-cell spatial gene expression with tissue morphology via explainable deep learning

Zhao, Y. — 2024-06-14

Deep learning models trained with spatial omics data uncover complex patterns and relationships among cells, genes, and proteins in a high-dimensional space. State-of-the-art in silico spatial multi-cell gene expression methods using histological images of tissue stained with hematoxylin and eosin (H&E) allow us to characterize cellular heterogeneity. We developed a vision transformer (ViT) framework to map histological signatures to spatial single-cell transcriptomic signatures, named SPiRiT. SPiRiT predicts single-cell spatial gene expression using the matched H&E image tiles of human breast cancer and whole mouse pup, evaluated by Xenium (10x Genomics) datasets. Importantly, SPiRiT incorporates rigorous strategies to ensure reproducibility and robustness of predictions and provides trustworthy interpretation through attention-based model explainability. SPiRiT model interpretation revealed the areas, and attention details it uses to predict gene expressions like marker genes in invasive cancer cells. In an apple-to-apple comparison with ST-Net, SPiRiT improved the predictive accuracy by 40%. These gene predictions and expression levels were highly consistent with the tumor region annotation. In summary, SPiRiT highlights the feasibility to infer spatial single-cell gene expression using tissue morphology in multiple-species.

Hair follicle-resident progenitor cells are a major cellular contributor to heterotopic subcutaneous ossifications in a mouse model of Albright hereditary osteodystrophy

McMullan, P. — 2024-06-21

Heterotopic ossifications (HOs) are the pathologic process by which bone inappropriately forms outside of the skeletal system. Despite HOs being a persistent clinical problem in the general population, there are no definitive strategies for their prevention and treatment due to a limited understanding of the cellular and molecular mechanisms contributing to lesion development. One disease in which the development of heterotopic subcutaneous ossifications (SCOs) leads to morbidity is Albright hereditary osteodystrophy (AHO). AHO is caused by heterozygous inactivation of GNAS, the gene that encodes the -stimulatory subunit (Gs) of G proteins. Previously, we had shown using our laboratorys AHO mouse model that SCOs develop around hair follicles (HFs). Here we show that SCO formation occurs due to inappropriate expansion and differentiation of HF-resident stem cells into osteoblasts. We also show in AHO patients and mice that Secreted Frizzled Related Protein 2 (SFRP2) expression is upregulated in regions of SCO formation and that elimination of Sfrp2 in male AHO mice exacerbates SCO development. These studies provide key insights into the cellular and molecular mechanisms contributing to SCO development and have implications for potential therapeutic modalities not only for AHO patients but also for patients suffering from HOs with other etiologies.

Structurally targeted mutagenesis identifies key residues supporting α-synuclein misfolding in multiple system atrophy

Reis, P. M. — 2024-07-09

Multiple system atrophy (MSA) and Parkinsons disease (PD) are caused by misfolded -synuclein spreading throughout the central nervous system. While familial PD is linked to several point mutations in -synuclein, there are no known mutations associated with MSA. Our previous work investigating differences in -synuclein misfolding between the two disorders showed that the familial PD mutation E46K inhibits replication of MSA prions both in vitro and in vivo, providing key evidence to support the hypothesis that -synuclein adopts unique strains in patients. Here, to further interrogate -synuclein misfolding, we engineered a panel of cell lines harboring both PD-linked and novel mutations designed to identify key residues that facilitate -synuclein misfolding in MSA. These data were paired with in silico analyses using Maestro software to predict the effect of each mutation on the ability of -synuclein to misfold into one of the reported MSA cryo-electron microscopy conformations. In many cases, our modeling accurately identified mutations that facilitated or inhibited MSA replication. However, Maestro was occasionally unable to predict the effect of a mutation on MSA propagation in vitro, demonstrating the challenge of using computational tools to investigate intrinsically disordered proteins. Finally, we used our cellular models to determine the mechanism underlying the E46K-driven inhibition of MSA replication, finding that the E46/K80 salt bridge is necessary to support -synuclein misfolding. Overall, our studies use a structure-based approach to investigate -synuclein misfolding, resulting in the creation of a powerful panel of cell lines that can be used to interrogate MSA strain biology.

Deliberative Behaviors and Prefrontal-Hippocampal Coupling are Disrupted in a Rat Model of Fetal Alcohol Spectrum Disorders

Rosenblum, H. L. — 2024-07-29

Fetal alcohol spectrum disorders (FASDs) are characterized by a range of physical, cognitive, and behavioral impairments. Determining how temporally specific alcohol exposure (AE) affects neural circuits is crucial to understanding the FASD phenotype. Third trimester AE can be modeled in rats by administering alcohol during the first two postnatal weeks, which damages the medial prefrontal cortex (mPFC), thalamic nucleus reuniens, and hippocampus (HPC), structures whose functional interactions are required for working memory and executive function. Therefore, we hypothesized that AE during this period would impair working memory, disrupt choice behaviors, and alter mPFC-HPC oscillatory synchrony. To test this hypothesis, we recorded local field potentials from the mPFC and dorsal HPC as AE and sham intubated (SI) rats performed a spatial working memory task in adulthood and implemented algorithms to detect vicarious trial and errors (VTEs), behaviors associated with deliberative decision- making. We found that, compared to the SI group, the AE group performed fewer VTEs and demonstrated a disturbed relationship between VTEs and choice outcomes, while spatial working memory was unimpaired. This behavioral disruption was accompanied by alterations to mPFC and HPC oscillatory activity in the theta and beta bands, respectively, and a reduced prevalence of mPFC-HPC synchronous events. When trained on multiple behavioral variables, a machine learning algorithm could accurately predict whether rats were in the AE or SI group, thus characterizing a potential phenotype following third trimester AE. Together, these findings indicate that third trimester AE disrupts mPFC-HPC oscillatory interactions and choice behaviors. Significance statementFetal alcohol spectrum disorders (FASDs) occur at an alarmingly high rate worldwide. Prenatal alcohol exposure leads to significant perturbations in brain circuitry that are accompanied by cognitive deficits, including disrupted executive functioning and working memory. These deficits stem from structural changes within several key brain regions including the prefrontal cortex, thalamic nucleus reuniens, and hippocampus. To better understand the cognitive deficits observed in FASD patients, we employed a rodent model of alcohol exposure during the third trimester, a period when these regions are especially vulnerable to alcohol-induced damage. We show that alcohol exposure disrupts choice behaviors and prefrontal-hippocampal functional connectivity during a working memory task, identifying the prefrontal-hippocampal network as a potential therapeutic target in FASD treatment.

MACanalyzeR: scRNA-seq Analysis Tool Reveals PPARγHI Lipid-Associated Macrophages Facilitate Thermogenic Expansion in BAT

Ninni, A. — 2024-07-29

Macrophages in brown adipose tissue (BAT) play a complex role in regulating its activity. However, the role of macrophages in regulating BAT activation/deactivation has not yet been comprehensively characterized. To elucidate this, we developed MACanalyzeR, a scRNAseq-based tool specifically designed to explore the macrophage features at molecular and metabolic level. MACanalyzeR was applied in scRNA-seq datasets obtained from BAT with thermogenic loss (db/db mice) and activation (High Fat Diet, HFD). Our computational approach revealed that macrophages accumulating in BAT upon these conditions resemble lipid-associated macrophages (LAMs) with foaming-like features. BAT LAMs also show a significant enrichment of genes associated with mitochondria and lysosomes. Interestingly, LAMs identified in BAT from HFD mice positively correlate with thermogenic genes and exhibit an enrichment in PPAR{gamma} signaling pathway, with an activated mitochondrial metabolism. Cell dynamic strategy, revealed that LAM with high Pparg expression levels (PpargHIGH) progressively accumulate during skeletal muscle regeneration, suggesting a potential role for this LAM subcluster in maintaining tissue homeostasis. Our findings suggest PpargHIGH LAMs as a subclass of macrophages potentially contributing in preserving tissue homeostasis associated with high energy demand conditions such as thermogenic and regenerative stimuli.

Immunodominant extracellular loops of Treponema pallidum FadL outer membrane proteins elicit antibodies with opsonic and growth-inhibitory activities

Delgado, K. N. — 2024-07-30

The global resurgence of syphilis has created a potent stimulus for vaccine development. To identify potentially protective antibodies (Abs) against Treponema pallidum (TPA), we used Pyrococcus furiosus thioredoxin (PfTrx) to display extracellular loops (ECLs) from three TPA outer membrane protein families (outer membrane factors for efflux pumps, eight-stranded {beta}-barrels, and FadLs) to assess their reactivity with immune rabbit serum (IRS). Five ECLs from the FadL orthologs TP0856, TP0858 and TP0865 were immunodominant. Rabbits and mice immunized with these five PfTrx constructs produced ECL-specific Abs that promoted opsonophagocytosis of TPA by rabbit peritoneal and murine bone marrow-derived macrophages at levels comparable to IRS and mouse syphilitic serum. ECL-specific rabbit and mouse Abs also impaired viability, motility, and cellular attachment of spirochetes during in vitro cultivation. The results support the use of ECL-based vaccines and suggest that ECL-specific Abs promote spirochete clearance via Fc receptor-independent as well as Fc receptor-dependent mechanisms. Author SummaryThe resurgence of syphilis emphasizes the critical need for vaccine development against Treponema pallidum (TPA). Research utilizing immune rabbit serum (IRS) suggests that an effective syphilis vaccine should induce "functional" antibodies (Abs) capable of enhancing the opsonophagocytosis of treponemes by activated macrophages. Structural models of TPA outer membrane proteins (OMPs), specifically the extracellular loops (ECLs), guided the identification of potential vaccine candidates. Antigenic analysis with IRS of individual ECLs from three TPA OMP families scaffolded onto Pyrococcus furiosus thioredoxin (PfTrx) revealed five FadL antigenic ECLs. Immunization with immunodominant ECL antigens elicited robust ECL-specific Abs, demonstrating functional opsonic activity in the opsonophagocytosis assays. Furthermore, these Abs effectively inhibited the growth inhibition of in vitro-cultivated TPA in both rabbit and mouse models. Our findings underscore the value of antigenic analysis in identifying promising TPA OMP ECL vaccine targets and highlight the multifaceted protective capacity of ECL Abs against TPA. This approach also extends to identifying potential OMP vaccinogens in other bacterial pathogens, offering valuable insights for broader vaccine development strategies.

Endothelial Immunosuppression in Atherosclerosis : Translational Control by Elavl1/HuR

Nicholas, S.-A. E. — 2024-08-04

Atherosclerotic plaques are defined by the accumulation of lipids and immune cells beneath the endothelium of the arterial intima. CD8 T cells are among the most abundant immune cell types in plaque, and conditions linked to their activation correlate with increased levels of cardiovascular disease. As lethal effectors of the immune response, CD8 T cell activation is suppressed at multiple levels. These checkpoints are critical in dampening autoimmune responses, and limiting damage in cardiovascular disease. Endothelial cells are well known for their role in recruiting CD8 T and other hematopoietic cells to low and disturbed flow (LDF) arterial regions that develop plaque, but whether they locally influence CD8 effector functions is unclear. Here, we show that endothelial cells can actively suppress CD8 T cell responses in settings of chronic plaque inflammation, but that this behavior is governed by expression of the RNA-binding protein Embryonic Lethal, Abnormal Vision-Like 1 (Elavl1). In response to immune cell recruitment in plaque, the endothelium dynamically shifts splicing of pre-mRNA and their translation to enhance expression of immune-regulatory proteins including C1q and CD27. This program is immuno-suppressive, and limited by Elavl1. We show this by Cdh5(PAC)-CreERT2-mediated deletion of Elavl1 (ECKO), and analysis of changes in translation by Translating Ribosome Affinity Purification (TRAP). In ECKO mice, the translational shift in chronic inflammation is enhanced, leading to increased ribosomal association of C1q components and other critical regulators of immune response and resulting in a [~]70% reduction in plaque CD8 T cells. CITE-seq analysis of the remaining plaque T cells shows that they exhibit lower levels of markers associated with T cell receptor (TCR) signaling, survival, and activation. To understand whether the immunosuppressive mechanism occurred through failed CD8 recruitment or local modulation of T cell responses, we used a novel in vitro co-culture system to show that ECKO endothelial cells suppress CD8 T cell expansion--even in the presence of wild-type myeloid antigen-presenting cells, antigen-specific CD8 T cells, and antigen. Despite the induction of C1q mRNA by T cell co-culture in both wild-type and ECKO endothelial cells, we find C1q protein abundantly expressed only in co-culture with ECKO cells. Together, our data define a novel immune-suppressive transition in the endothelium, reminiscent of the transition of T cells to T-regs, and demonstrate the regulation of this process by Elavl1.

The Role of Sex in Atherosclerotic Inflammation and Lipid-Handling Dysfunction

Liu, P. Z. — 2024-08-26

Residual risk of cardiovascular events remains despite treatments that effectively lower cholesterol levels and hypertension, suggesting that there must be more variables to consider in atherosclerosis treatment. Several studies have suggested sex1,2,3 and inflammation4,5 as important variables. However, a cross-cohort analysis of sex and risk factors like inflammation and lipid-handling dysfunction is needed to strengthen their connection to atherosclerosis. By using blood transcriptomic profiles of 391 male and female participants, this study revealed that inflammation and lipid-handling dysfunction have sex-specific roles in atherosclerosis. Transcriptomics of 391 human blood samples with varying degrees of atherosclerosis were used to identify sex-specific changes in immune response and lipid-handling in circulating blood cells. Preliminary analyses of both FPKM and normalized counts datasets showed that inflammatory pathway activation and enrichment increased as atherosclerotic disease severity increased across all sexes. Analysis of sex-specific differentially expressed genes (DEGs) using IPAs Canonical Pathways showed that severely impacted females had more enriched inflammatory pathways than severely impacted males. Further cross-cohort analysis of sex-specific inflammation and lipid-handling dysfunction was performed using AtheroSpectrum, a single-sample macrophage annotation tool. AtheroSpectrum confirmed that inflammation was more critical to female atherogenesis and revealed that lipid-handling dysfunction was more critical to male atherogenesis. Our study underscored the importance of inflammation and sex as variables to consider in atherosclerosis treatment, suggesting that treatment should target inflammation and consider sex. Our findings may be used for generating a model to predict atherosclerosis risk based on key DEGs, pathways, sex, and other clinical parameters when available.

Track-A-Worm 2.0: A Software Suite for Quantifying Properties of C. elegans Locomotion, Bending, Sleep, and Action Potentials

Vedantham, K. — 2024-09-15

Comparative analyses of locomotor behavior and cellular electrical properties between wild-type and mutant C. elegans are crucial for exploring the gene basis of behaviors and the underlying cellular mechanisms. Although many tools have been developed by research labs and companies, their application is often hindered by implementation difficulties or lack of features specifically suited for C. elegans. Track-A-Worm 2.0 addresses these challenges with three key components: WormTracker, SleepTracker, and Action Potential (AP) Analyzer. WormTracker accurately quantifies a comprehensive set of locomotor and body bending metrics, reliably distinguish between the ventral and dorsal sides, continuously tracks the animal using a motorized stage, and seamlessly integrates external devices, such as a light source for optogenetic stimulation. SleepTracker detects and quantifies sleep-like behavior in freely moving animals. AP Analyzer assesses the resting membrane potential, afterhyperpolarization level, and various AP properties, including threshold, amplitude, mid-peak width, rise and decay times, and maximum and minimum slopes. Importantly, it addresses the challenge of AP threshold quantification posed by the absence of a pre-upstroke inflection point. Track-A-Worm 2.0 is potentially a valuable tool for many C. elegans research labs due to its powerful functionality and ease of implementation.

Real-time affinity measurements of proteins synthesized in cell-free lysate using fluorescence correlation spectroscopy

Liu, C. — 2024-09-30

Rapid, high throughput measurements of biomolecular interactions are essential across medicine and bioscience. Traditional methods for affinity-screening proteins require a long and costly process involving cell-based expression, purification, and titration of multiple concentrations to arrive at a binding curve. In contrast, we have developed a fast and simple approach that yields a wealth of information about the expression of the protein and its binding characteristics, all in a "one-pot reaction" and done in under several hours without the need for protein purification. The method uses cell-free protein synthesis to produce the protein of interest in the presence of its binding partner, while simultaneously using fluorescence correlation spectroscopy (FCS) to measure the increasing concentration of the protein and its binding to the binding partner. We characterize the sensitivity limits of this method by measuring the binding between the green fluorescent protein (GFP) and a low picomolar-affinity anti-GFP antibody and found that we can quantify KDs down to the high picomolar to low-nanomolar range. We further demonstrate the method in a potentially ultrahigh-throughput sample format, in which FCS measurements are collected inside microcapsules. This work lays the foundation for a platform aimed at production and in situ affinity screening of thousands of different proteins.

TET2 contributes to gluconeogenesis and pathology of type 2 diabetes

Zhang, X. — 2024-10-01

The control of gluconeogenesis is critical for glucose homeostasis and the pathology of type 2 diabetes (T2D). Here, we uncover a novel function of TET2 in the regulation of gluconeogenesis. In mice, both fasting and a high-fat diet (HFD) stimulate the expression of TET2, and TET2 knockout impairs glucose production. Mechanistically, FBP1, a rate-limiting enzyme in gluconeogenesis, is positively regulated by TET2 in liver cells. TET2 is recruited by HNF4, contributing to the demethylation of FBP1 promoter and activating its expression in response to glucagon stimulation. Moreover, metformin treatment increases the phosphorylation of HNF4 on Ser313, which prevents its interaction with TET2, thereby decreasing the expression level of FBP1 and ameliorating the pathology of T2D. Collectively, we identify an HNF4-TET2-FBP1 axis in the control of gluconeogenesis, which contributes to the therapeutic effect of metformin on T2D and provides a potential target for the clinical treatment of T2D.

Impact of ligand binding on VEGFR1, VEGFR2, and NRP1 localization in human endothelial cells

Sarabipour, S. — 2024-10-01

The vascular endothelial growth factor receptors (VEGFRs) bind to cognate ligands to facilitate signaling pathways critical for angiogenesis, the growth of new capillaries from existing vasculature. Intracellular trafficking regulates the availability of receptors on the cell surface to bind ligands, which regulate activation, and the movement of activated receptors between the surface and intracellular pools, where they can initiate different signaling pathways. Using experimental data and computational modeling, we recently demonstrated and quantified the differential trafficking of three VEGF receptors, VEGFR1, VEGFR2, and coreceptor Neuropilin-1 (NRP1). Here, we expand that approach to quantify how the binding of different VEGF ligands alters the trafficking of these VEGF receptors and demonstrate the consequences of receptor localization and ligand binding on the localization and dynamics of signal initiation complexes. We include simulations of four different splice isoforms of VEGF-A and PLGF, each of which binds to different combinations of the VEGF receptors, and we use new experimental data for two of these ligands to parameterize and validate our model. We show that VEGFR2 trafficking is altered in response to ligand binding, but that trafficking of VEGFR1 is not; we also show that the altered trafficking can be explained by a single mechanistic process, increased internalization of the VEGFR2 receptor when bound to ligand; other processes are unaffected. We further show that even though the canonical view of receptor tyrosine kinases is of activation on the cell surface, most of the ligand-receptor complexes for both VEGFR1 and VEGFR2 are intracellular. We also explore the competition between the receptors for ligand binding, the so-called decoy effect, and show that while in vitro on the cell surface minimal such effect would be observed, inside the cell the effect can be substantial and may influence signaling. We term this location dependence the reservoir effect as the size of the local ligand reservoir (large outside the cell, small inside the cell) plays an integral role in the receptor-receptor competition. These results expand our understanding of receptor-ligand trafficking dynamics and are critical for the design of therapeutic agents to regulate ligand availability to VEGFR1 and hence VEGF receptor signaling in angiogenesis.

The Unique Role of Intracellular Perinuclear β-Adrenergic Receptors in defining Signaling Compartmentation and Pathological Cardiac Remodeling

Turcotte, M. G. — 2024-10-03

The {beta}-adrenergic receptor is a prototypical G-protein coupled receptor that initiates signaling from the plasma membrane. However, active receptors have been detected within intracellular compartments. The functional significance of these intracellular receptors remains unclear, including whether they regulate distinct cellular processes or function independently of plasma membrane receptors. We show using live cell imaging of primary cardiomyocytes that {beta}-adrenergic receptors localized to Golgi apparatus opposing the outer nuclear membrane are sufficient and necessary for the stimulation of cAMP and calcium signaling within a nanometer scale compartment independent of receptors at other sites. Using compartment-specific activators and inhibitors, we show Golgi {beta}-adrenergic receptors associated with the scaffold protein AKAP6{beta} and the outer nuclear membrane protein nesprin-1 are responsible for pathological gene transcription and the induction of cardiomyocyte hypertrophy. The functional significance of Golgi-localized receptors is demonstrated in mice models of cardiomyopathy, providing proof-of-concept for a compartment-specific therapeutic intervention in Dilated Cardiomyopathy.

LPS binding caspase activation and recruitment domains (CARDs) are bipartite lipid binding modules

Cao, A. B. — 2024-10-11

Caspase-11 is an innate immune pattern recognition receptor (PRR) that detects cytosolic bacterial lipopolysaccharides (LPS) through its caspase activation and recruitment domain (CARD), triggering inflammatory cell death known as pyroptosis. Caspase-11 also detects eukaryotic (i.e. self) lipids. This observation raises the question of whether common or distinct mechanisms govern the interactions with self and nonself lipids. In this study, using biochemical, computational, and cell-based assays, we report that the caspase-11 CARD functions as a bipartite lipid-binding module. Distinct regions within the CARD bind to phosphate groups and long acyl chains of self and nonself lipids. Self-lipid binding capability is conserved across numerous caspase-11 homologs and orthologs. The symmetry in self and nonself lipid detection mechanisms enabled us to engineer an LPS-binding domain de novo, using an ancestral CARD-like domain present in the fish Amphilophus citrinellus. These findings offer critical insights into the molecular basis of LPS recognition by caspase-11 and highlight the fundamental and likely inseparable relationship between self and nonself discrimination.

Decoding Spatial Tissue Architecture: A Scalable Bayesian Topic Model for Multiplexed Imaging Analysis

Peng, X. — 2024-10-13

Recent progress in multiplexed tissue imaging is advancing the study of tumor microenvironments to enhance our understanding of treatment response and disease progression. Cellular neighborhood analysis is a popular computational approach for these complex image data. Despite its popularity, there are significant challenges, including high computational demands that limit feasibility for largescale applications and the lack of a principled strategy for integrative analysis across images. This absence hampers the precise and consistent identification of spatial features and tracking of their dynamics over disease progression. To overcome these challenges, we introduce SpatialTopic, a spatial topic model designed to decode high-level spatial architecture across multiplexed tissue images. SpatialTopic integrates both cell type and spatial information within a topic modelling framework, originally developed for natural language processing and adapted for computer vision. Spatial information is incorporated into the flexible design of documents, representing densely overlapping regions in images. We employ an efficient collapsed Gibbs sampling algorithm for model inference. We benchmarked the performance against five state-of-the-art algorithms through various case studies using different single-cell spatial transcriptomic and proteomic imaging platforms across different tissue types. We show that SpatialTopic is highly scalable on large-scale image datasets with millions of cells, along with high precision and interpretability. Our findings demonstrate that SpatialTopic consistently identifies biologically and clinically significant spatial "topics" such as tertiary lymphoid structures (TLSs) and tracks dynamic changes in spatial features over disease progression. Its computational efficiency and broad applicability across various molecular imaging platforms will enhance the analysis of large-scale tissue imaging datasets.

DNA polymerase η is regulated by mutually exclusive mono-ubiquitination and mono-NEDDylation

Moreno, N. C. — 2024-10-13

DNA polymerase eta (Pol {eta}) is a Y-family translesion polymerase responsible for synthesizing new DNA across UV-damaged templates. It is recruited to replication forks following mono-ubiquitination of the PCNA DNA clamp. This interaction is mediated by PCNA-interacting protein (PIP) motifs within Pol {eta}, as well as by its C-terminal ubiquitin-binding zinc finger (UBZ) domain. Previous work has suggested that Pol {eta} itself is mono-ubiquitinated at four C-terminal lysine residues, which is dependent on prior ubiquitin-binding by its UBZ domain. Here, we show that Pol {eta} can be modified at the same lysine residues by the ubiquitin-like protein, NEDD8. Like ubiquitination, this modification is driven by non-covalent interactions between NEDD8 and the UBZ domain. While only a small proportion of Pol {eta} is mono-NEDDylated under normal conditions, these levels rapidly increase by inhibiting the COP9 signalosome, suggesting that mono-NEDDylation is maintained under strong negative regulation. Finally, we provide data to support that mono-ubiquitination is important for Pol {eta} foci formation and suggest that NEDDylation disrupts this process. These results reveal a new mechanism of Pol {eta} regulation by ubiquitin-like proteins.

The Integrated Stress Response Suppresses PINK1-dependent Mitophagy by Preserving Mitochondrial Import Efficiency

Yang, M. — 2024-10-17

Mitophagy is crucial for maintaining mitochondrial health, but how its levels adjust to different stress conditions remains unclear. In this study, we investigated the role of the DELE1-HRI axis of integrated stress response (ISR) in regulating mitophagy, a key mitochondrial stress pathway. Our findings show that the ISR suppresses mitophagy under non-depolarizing mitochondrial stress by positively regulating mitochondrial protein import, independent of ATF4 activation. Mitochondrial protein import is regulated by the rate of protein synthesis under both depolarizing and non-depolarizing stress. Without ISR, increased protein synthesis overwhelms the mitochondrial import machinery, reducing its efficiency. Under depolarizing stress, mitochondrial import is heavily impaired even with active ISR, leading to significant PINK1 accumulation. In contrast, non-depolarizing stress allows more efficient protein import in the presence of ISR, resulting in lower mitophagy. Without ISR, mitochondrial protein import becomes severely compromised, causing PINK1 accumulation to reach the threshold necessary to trigger mitophagy. These findings reveal a novel link between ISR-regulated protein synthesis, mitochondrial import, and mitophagy, offering potential therapeutic targets for diseases associated with mitochondrial dysfunction.

Development and utilization of Treponema pallidum expressing green fluorescent protein to study spirochete-host interactions and antibody-mediated clearance: expanding the toolbox for syphilis research.

Delgado, K. N. — 2024-10-21

Syphilis is a sexually transmitted infection caused by the highly invasive and immunoevasive spirochetal pathogen Treponema pallidum subsp. pallidum (TPA). Untreated syphilis can lead to infection of multiple organ systems, including the central nervous system. The alarming increase in syphilis cases globally underscores the importance of developing novel strategies to understand the complexities of syphilis pathogenesis. In this study, we took advantage of recent advances in in vitro cultivation and genetic manipulation of syphilis spirochetes to engineer a TPA strain that constitutively expresses green fluorescent protein (GFP). GFP+ TPA grew identically to the Nichols parent strain in vitro and exhibited wild-type infectivity in the rabbit model. We then used the GFP+ strain to visualize TPA interactions with host cells during co-cultivation in vitro, within infected rabbit testes, and following opsonophagocytosis by murine bone marrow-derived macrophages. Development of fluorescent strain also enabled us to develop a flow cytometric-based assay to assess antibody-mediated damage to the spirochetes fragile outer membrane (OM), demonstrating dose-dependent growth inhibition and OM disruption in vitro. Notably, we observed greater OM disruption of GFP+ TPA with sera from immune rabbits infected with the TPA Nichols strain compared to sera generated against the genetically distinct SS14 strain. These latter findings highlight the importance of OM protein-specific antibody responses for clearance of TPA during syphilitic infection. The availability of fluorescent TPA strains paves the way for future studies investigating spirochete-host interactions as well as functional characterization of antibodies directed treponemal OM proteins, the presumptive targets for protective immunity. ImportanceSyphilis, a sexually transmitted infection caused by Treponema pallidum (TPA), remains a pressing threat to global public health. TPA has a remarkable and still poorly understood ability to disseminate rapidly from the site of inoculation and establish persistent infection throughout the body. Recent advances in in vitro cultivation and genetic manipulation of syphilis spirochetes enabled the development of fluorescent TPA. In the study, we generated and characterized an infectious TPA strain that constitutively expresses green fluorescent protein and used this strain to visualize interaction of TPA with host cells and functionally characterize antibodies directed against treponemal outer membrane proteins. Most notably, we assessed the ability of surface-bound antibodies to inhibit growth of TPA in vitro and/or disrupt the spirochetes fragile outer membrane. Fluorescent TPA strains provide a powerful new tool for elucidating host-pathogen interactions that enable the syphilis spirochete to establish infection and persistent long-term within its obligate human host.

Ethanolamine-induced assembly of microcompartments is required for Fusobacterium nucleatum virulence

Franklin, D. — 2024-11-11

Many bacteria metabolize ethanolamine as a nutrient source through cytoplasmic organelles named bacterial microcompartments (BMCs). Here we investigated the molecular assembly, regulation, and function of BMCs in Fusobacterium nucleatum - a Gram-negative oral pathobiont that is associated with adverse pregnancy outcomes. The F. nucleatum genome harbors a conserved ethanolamine utilization (eut) locus with 21 genes that encode several putative BMC shell proteins and a two-component signal transduction system (TCS), in addition to the enzymes for ethanolamine transport and catabolism. We show that the expression of most of these genes as well as BMC formation is highly increased in wild type fusobacteria when cultured in the presence of ethanolamine as a nutrient source. Deletion of the response regulator EutV eliminated this induction of eut mRNAs and BMCs, thus demonstrating that BMC formation is transcriptionally regulated by the TCS EutV-EutW in response to ethanolamine. Mass spectrometry of isolated BMCs unveiled the identity of the constituent proteins EutL, EutM1, EutM2, and EutN. Consistent with the role of these proteins in BMC assembly and metabolism, deletion of eutN, eutL/eutM1/eutM2, or eutL/eutM1/eutM2/eutN not only affected BMC formation, but also ethanolamine utilization, causing cell growth defects with ethanolamine as nutrient. BMCs also assembled in fusobacteria cultured with placental cells or the culture media, a process that is dependent on the BMC shell proteins. Significantly, we show that the eutN mutant is defective in inducing preterm birth in a mouse model. Together, these results establish that BMC-mediated metabolism of ethanolamine is critical for fusobacterial virulence. IMPORTANCEThe oral anaerobe Fusobacterium nucleatum can spread to distal internal organs, such as the colon and placenta, and thereby promote the development of colorectal cancer and induce preterm birth, respectively. Yet, how this opportunistic pathogen adapts to the various metabolically distinct host cellular niches remains poorly understood. We demonstrated here that this microbe assembles specialized metabolic organelles, termed bacterial microcompartments (BMCs), to utilize environmental ethanolamine (EA) as a key environmental nutrient source. The formation of F. nucleatum BMCs, containing BMC shell proteins EutLM1M2N, is controlled by a two-component system, EutV-EutW, responsive to EA. Significantly, this ability of F. nucleatum to form BMCs in response to EA is crucial for its pathogenicity evidenced by the fact that the genetic disruption of BMC formation reduces fusobacterial virulence in a mouse model of preterm birth.

Lactate potentiates NMDA receptor currents via an intracellular redox mechanism targeting cysteines in the C-terminal domain of GluN2B subunits: implications for synaptic plasticity

Fiumelli, H. — 2024-11-21

SummaryThrough the Astrocyte Neuron Lactate Shuttle, astrocyte-derived lactate fuels the high-energy demands of neurons and acts as a signaling molecule, promoting synaptic plasticity and memory consolidation. Lactate regulates neuronal excitability and modulates the expression of genes related to synaptic plasticity and neuroprotection, but the molecular mode for these signaling actions is uncertain. Using patch-clamp recordings in cultured cortical neurons, we found that lactate enhances both the amplitude and the inactivation time constant of NMDA receptor currents (INMDAR) evoked by brief applications of glutamate and glycine. Not reproduced by HCAR1 agonists, this modulation depends on monocarboxylate transporters and lactate dehydrogenase, indicating the requirement for lactate entry and metabolic conversion into pyruvate and NADH formation within neurons. Disruption of intracellular calcium dynamics or inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a NMDAR-associated kinase linking Ca2+ signal to long-term potentiation (LTP), significantly diminishes the effects of lactate on INMDAR. We identified two redox-sensitive cysteine- containing sequences in the intrinsically disordered intracellular C-terminal domain of the GluN2B subunit that play a role in the potentiation of NMDAR by lactate. In a compelling set of experiments using HEK cells, we observed that the presence of functional CaMKII and GluN2B-containing NMDARs is necessary for the lactate-enhancing effects. Mutations in GluN2B that prevent CaMKII binding or redox regulation via cysteines abrogate the modulatory action of lactate. Immunoprecipitation experiments in neurons attest that lactate increases the association between CaMKII and GluN2B. This interaction is crucial for the potentiation of INMDAR amplitude by lactate. Proximity ligation assays between GluN2B and the postsynaptic density marker PSD-95 revealed that lactate induced an accumulation of GluN2B in dendritic spines, an effect that was prevented by a CaMKII peptide inhibitor. These results highlight a mechanistic pathway whereby lactate boosts NMDAR function through intracellular metabolic conversion and redox-sensitive interactions requiring CaMKII, establishing a link between astrocyte metabolism and synaptic modulation in neurons.

Cardiac Applications of CRISPR/AAV-Mediated Precise Genome Editing

Zheng, Y. — 2024-12-04

The ability to efficiently make precise genome edits in somatic tissues will have profound implications for gene therapy and basic science. CRISPR/Cas9 mediated homology-directed repair (HDR) is one approach that is commonly used to achieve precise and efficient editing in cultured cells. Previously, we developed a platform capable of delivering CRISPR/Cas9 gRNAs and donor templates via adeno-associated virus to induce HDR (CASAAV-HDR). We demonstrated that CASAAV-HDR is capable of creating precise genome edits in vivo within mouse cardiomyocytes at the neonatal and adult stages. Here, we report several applications of CASAAV-HDR in cardiomyocytes. First, we show the utility of CASAAV-HDR for disease modeling applications by using CASAAV-HDR to create and precisely tag two pathological variants of the titin gene observed in cardiomyopathy patients. We used this approach to monitor the cellular localization of the variants, resulting in mechanistic insights into their pathological functions. Next, we utilized CASAAV-HDR to create another mutation associated with human cardiomyopathy, arginine 14 deletion (R14Del) within the N-terminus of Phospholamban (PLN). We assessed the localization of PLN-R14Del and quantified cardiomyocyte phenotypes associated with cardiomyopathy, including cell morphology, activation of PLN via phosphorylation, and calcium handling. After demonstrating CASAAV-HDR utility for disease modeling we next tested its utility for functional genomics, by targeted genomic insertion of a library of enhancers for a massively parallel reporter assay (MPRA). We show that MPRAs with genomically integrated enhancers are feasible, and can yield superior assay sensitivity compared to tests of the same enhancers in an AAV/episomal context. Collectively, our study showcases multiple applications for in vivo precise editing of cardiomyocyte genomes via CASAAV-HDR.

Bridging molecular to cellular scales for models of membrane receptor signaling

Peterson, K. J. — 2024-12-05

Biochemical interactions at membranes are the starting points for cell signaling networks. But bimolecular reaction kinetics are difficult to experimentally measure on 2-dimensional membranes and are usually measured in volumetric in vitro assays. Membrane tethering produces confinement and steric effects that will significantly impact binding rates in ways that are not readily estimated from volumetric measurements. Also, there are situations when 2D reactions do not conform to simple mass action kinetics. Here we show how highly coarse-grained molecular simulations using the SpringSaLaD software can be used to estimate membrane-tethered rate constants from experimentally determined volumetric kinetics. The approach is validated using an analytical solution for dimerization of binding sites anchored via stiff linkers. This approach can provide 2-dimensional bimolecular rate constants to parameterize cell-scale models of receptor-mediated signaling. We explore how factors such as molecular reach, steric effects, disordered domains, local concentration and diffusion affect the kinetics of binding. We also develop a general scheme to assess whether simple mass action rate constants can be applied for a given scenario, taking into account the diffusivity of the membrane anchors and tethered binding sites, the initial membrane densities of the reactants and the desired level of completion for the fitted rate constant. We then apply our approach to epidermal growth factor receptor (EGFR) mediated activation of the membrane-bound small GTPase Ras. The analysis reveals how binding of Ras to the allosteric site of SOS, a guanine nucleotide exchange factor (GEF) that is recruited to EGFR, significantly accelerates Ras binding to the SOS catalytic site. A small biochemical network model parametrized with the derived 2D rate constants shows how recruitment of SOS via EGFR can significantly enhance Ras activation. SIGNIFICANCE STATEMENTIn cell signaling, the activation of a surface receptor leads to a cascade of intracellular biochemical events. Many protein interactions occur near the inner plasma membrane surface. However, accurate rate parameters for these steps in models of signaling are rarely available because membrane-tethered reaction kinetics are difficult to experimentally measure. Here, we use a highly coarse-grained molecular simulator to model the kinetics of reactions between binding sites that are tethered to a membrane. We can fit these simulation outputs with 2-dimensional rate laws to obtain rate constants that can be used to build complex models of cell signaling. The derived rate constants can also be analyzed to understand the key biophysical features controlling the kinetics of bimolecular membrane reactions.

Sound-Evoked Plasticity Differentiates Tinnitus from Non-Tinnitus Mice

Fabrizio-Stover, E. M. — 2024-12-21

Tinnitus is the perception of non-meaningful sound in the absence of external stimuli. Although tinnitus behavior in animal models is associated with altered central nervous system activity, it is not currently possible to identify tinnitus using neuronal activity alone. In the mouse inferior colliculus (IC), a subpopulation of neurons demonstrates a sustained increase in spontaneous activity after a long-duration sound (LDS). Here, we use the "LDS test" to reveal tinnitus-specific differences in sound-evoked plasticity through IC extracellular recordings and the auditory brainstem response (ABRLDS) in CBA/CaJ mice after sound exposure and behavioral tinnitus assessment. Sound-exposed mice showed stronger and shorter tone-evoked responses in the IC compared to unexposed controls, but these differences were not strong predictors of tinnitus. In contrast, in the LDS test, non-tinnitus mice had a significantly stronger suppression in tone-evoked spike rate compared to tinnitus and unexposed control mice. ABR peak amplitudes also revealed robust differences between tinnitus and non-tinnitus mice, with ABR peaks from non-tinnitus mice exhibiting significantly stronger suppression in the LDS test compared to tinnitus and control mice. No significant differences were seen between cohorts in ABR amplitude, latency, wave V:I ratio, wave V:III ratio, I-V intra-peak latency, and I-VI intra-peak latency. We found high-frequency tone stimuli better suited to reveal tinnitus-specific differences for both extracellular IC and ABR recordings. We successfully used the LDS test to demonstrate that tinnitus-specific differences in sound-evoked plasticity can be shown using both multi-unit near-field recordings in the IC and non-invasive far-field recordings, providing a foundation for future electrophysiological research into the causes and treatment of tinnitus.

Bovine Formative Embryonic Stem Cell Plasticity in Embryonic and Extraembryonic Differentiation

Su, Y. — 2024-12-29

Bovine embryonic stem cells (bESCs) can greatly enhance understanding of bovine embryonic development and applications for disease-resistance, biomedical and zoonotic pre-clinical models. However, formative bESCs with distinct morphology and complete differentiation capacity are still unreported. We document here the generation of bESCs which are pluripotent both in vitro and in vivo, and efficiently converted into neural progenitor cells (NPCs) and primordial germ cell-like cells (PGCLCs) by direct differentiation. These cells exhibited distinct metabolic features from human and mouse ESCs and early embryos. Formative bESCs contributed to a wide range of cell types within embryonic and extraembryonic tissues after aggregating with mouse and bovine embryos. The establishment of bovine formative ESCs with dual developmental plasticity represents a milestone for agricultural biotechnology and decoding the underlying mechanism of bona fide bovine pluripotency. Summary statementGenerating bovine ESCs would greatly enhance understanding of bovine embryonic development and bioengineering of cattle for disease-resistance, biomedical and zoonotic pre-clinical models.

A direct interaction between the RNA-binding proteins Staufen and Tm1-I/C regulates oskar mRNP composition and transport

Gaber, T. — 2024-12-30

In the Drosophila female germline, oskar messenger RNA is transported on microtubules from the nurse cells to the posterior pole of the oocyte, where it is translated. Transport of oskar transcripts from the nurse cells into the oocyte requires dynein, while localization of the mRNAs within the oocyte to the posterior pole is dependent upon kinesin-1. Staufen, a dsRNA-binding protein, has been shown to bind the oskar mRNA transport complex in the oocyte and inactivate dynein; however, it remains unclear how kinesin is activated. Here, using surface plasmon resonance, nuclear magnetic resonance spectroscopy and RNA imaging within egg chambers, we demonstrate that Staufen directly interacts with Tm1, a non-canonical kinesin adaptor. This work provides a molecular explanation for the previously unclear role of Staufen in oskar mRNA localization.

From spots to stripes: Evolution of pigmentation patterns in monkeyflowers via modulation of a reaction-diffusion system and its prepatterns

Liang, M. — 2025-01-11

The reaction-diffusion (RD) system is widely assumed to account for many complex, self- organized pigmentation patterns in natural organisms. However, the specific configurations of such RD networks and how RD systems interact with positional information (i.e., prepatterns) that may specify the initiation conditions for the RD operation remain largely unknown. Here, we introduced a three-substance RD system underlying the formation of repetitive pigment spots and stripes in Mimulus flowers. It consists of an R2R3-MYB activator (NEGAN), an R3-MYB inhibitor (RTO), and a coactivator represented by two paralogous bHLH proteins. Through fine- scale genetic analyses, transgenic experiments, and computer simulations, we identified the causal loci contributing to the evolutionary transition from sparsely dispersed spots to longitudinal stripes. Genetic changes at these loci modulate the prepatterns of the activator and coactivator expression and the promoter activities of the inhibitor and one of the coactivator paralogs. Our findings highlight the importance of prepatterns towards a realistic description of RD systems in natural organisms, and reveal the genetic mechanism generating pattern variation through modulation of the kinetics of the RD system and its prepatterns.

Sequence variability of BamA and FadL candidate vaccinogens suggests divergent evolutionary paths of Treponema pallidum outer membrane proteins

Bettin, E. B. — 2025-04-23

Knowledge of Treponema pallidum subspecies pallidum (TPA) outer membrane protein (OMP) sequence variability is essential for understanding spirochete proliferation within endemic populations as well as design of a globally effective syphilis vaccine. Our group has identified extracellular loops (ECLs) of TPA BamA (TP0326) and members of the FadL family (TP0548, TP0856, TP0858, TP0859, and TP0865) as potential components of a multivalent vaccine cocktail. As part of a consortium to explore TPA strain diversity, we mapped the variability of BamA and FadL orthologs in 186 TPA strains from Malawi, China and Colombia onto predicted 3D structures. The 186 genomes fell into eight subclades (five Nichols-, three SS14-lineage) with substantial geographic restriction. Single nucleotide variants accounted for the large majority of proteoforms, with variability notably higher within the Nichols-lineage strains. Most mutations were in regions of the proteins predicted to be extracellular and harboring B cell epitopes. We observed a striking difference in the degree of variability between the six OMPs, suggesting that these proteins are following divergent evolutionary paths. Concatenation of OMP sequences recapitulated the phylogenetic structure of the TPA strains, effectively segregating within clades and largely clustering by subclades. Lastly, we noted that BamA and FadL candidate ECL vaccinogens previously shown to elicit antibodies that kill treponemes during in vitro cultivation are well conserved. Taken as a whole, our study establishes a structural-phylogenetic approach for analyzing the forces shaping the host-pathogen interface in syphilis within endemic populations while informing selection of vaccine targets. IMPORTANCESyphilis remains a major global health concern, reinforcing the need for a safe and effective vaccine. Understanding the variability of TPA OMPs is essential for tracking pathogen evolution and informing vaccine design. Here, we analyzed the variability of six TPA OMPs in 186 strains from Malawi, China, and Colombia, identifying protein-specific evolutionary patterns. Most mutations were localized in extracellular regions and, notably, appeared to correlate with the phylogenetic structure of TPA. Despite OMP heterogeneity, several candidate vaccinogens remained highly conserved, reinforcing their potential as globally effective vaccine targets. Our study establishes a structural-phylogenetic framework for dissecting the forces shaping the host-spirochete interface within endemic populations and provides a foundation for designing a globally effective syphilis vaccine.

Mechanistic and evolutionary insights into a family of aminoacyl-tRNA deacylases that protects against canavanine toxicity

Maldonado, J. S. — 2025-05-04

Aminoacyl-tRNA deacylases safeguard the accurate translation of the genetic code by hydrolyzing incorrectly synthesized aminoacyl-tRNAs. Canavanyl-tRNA deacylase (CtdA) was recently shown to protect cells against the toxicity of the non-proteinogenic amino acid canavanine, which is synthesized and accumulated by various plants. In most organisms, canavanine is ligated to tRNAArg, causing translation of arginine codons with canavanine. CtdA prevents canavanine toxicity by hydrolyzing canavanyl-tRNAArg. Here, we investigated the function, structure, substrate specificity, phylogenetic distribution, and evolution of CtdA. We show that CtdA is essential to prevent canavanine cytotoxicity in Salmonella enterica, and its heterologous expression can also rescue Escherichia coli. By determining the structure of CtdA, we identified its putative binding pocket and residues that modulate enzymatic activity and specificity. We also found that CtdA displays a relaxed specificity for the aminoacyl moiety substrate as it hydrolyzes arginyl-tRNAArg. Finally, we showed that despite their structural homology, CtdA and the aminoacyl-tRNA hydrolytic domain of phenylalanyl-tRNA synthetase are functionally and evolutionarily divergent. Collectively, these results substantially expand our understanding of the CtdA family, providing new insights into its structure, function, and evolution. Moreover, this work highlights the diverse mechanisms unique to each organism to ensure faithful translation of the genetic code.

A human and mouse subpopulation of senescent β-cells induces pathologic dysfunction through targetable paracrine signaling

Iwasaki, K. — 2025-05-07

Cellular senescence is a stress response mechanism marked by irreversible growth arrest, upregulation of antiapoptotic pathways, loss of cellular function, and remodelling of the cellular secretory profile. In both humans and mice, pancreatic {beta}-cells undergo senescence with age and insulin resistance. Targeted removal of senescent cells in mouse models of diabetes improves glucose homeostasis, demonstrating the role {beta}-cell senescence in diabetes progression. In contrast, {beta}-cell senescence also promotes immune surveillance, promoting {beta}-cell survival and function. Thus, a better understanding of senescent cells phenotypic and functional heterogeneity is needed to develop effective therapeutic strategies. Herein, we show that subpopulations of senescent {beta}-cells in mice and humans, which were identified through the expression of Cdkn1a (encoding p21Cip1) and Cdkn2a (encoding p16Ink4a) by single-cell RNA sequencing (scRNA-seq), flow cytometry, spatial transcriptomics, and spatial proteomics, exhibit distinct transcriptional and functional identities. The predominant senescent {beta}-cell subpopulation expressed Cdkn1a and was characterized by a lack of glucose responsiveness, high basal insulin secretion, and transcription of canonical SASP factors. The SASP of Cdkn1a-expressing {beta}-cells had non-cell autonomous effects on neighbouring cells. A subset of four SASP factors from Cdkn1a+ cells was sufficient to induce secondary senescence and {beta}-cell dysfunction in vitro. JAK inhibitors (JAK1/2 and JAK1/3) counteracted secondary senescence induction and restored {beta}-cell function in high-fat diet-fed mice and human islets from donors with or without type 2 diabetes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/648438v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@9b8addorg.highwire.dtl.DTLVardef@1b9c4eborg.highwire.dtl.DTLVardef@12f2964org.highwire.dtl.DTLVardef@1468297_HPS_FORMAT_FIGEXP M_FIG C_FIG

Targeting of CIP4-Calcineurin Signalosomes Improves Cardiac Structure and Function After Myocardial Infarction

Samuelsson, A.-M. — 2025-05-15

BackgroundCalcineurin in a pleiotropic signaling enzyme that promotes pathological cardiac remodeling but also cardioprotection in ischemia-reperfusion injury. In addition, calcineurin inhibitors are immunosuppressants. This pleiotropy has precluded the use of calcineurin inhibitors as treatments for heart failure. Cdc42-interacting protein 4 (CIP4/TRIP10) is an endosomal scaffold protein that organizes a calcium and calcineurin A{beta}2 (CaNA{beta}2) signaling compartment activated by G-protein coupled receptors independently of contractile calcium. CIP4 binds CaNA{beta}2 via the CaNA{beta}-specific N-terminal polyproline (PP) domain. We previously showed that targeting of CIP4-CaNA{beta}2 signalosomes inhibited pathological cardiac hypertrophy and the development of heart failure induced by chronic pressure overload in mice. It is unknown whether CIP4-CaNA{beta}2 signalosomes contribute to cardioprotection and/or cardiac remodeling in ischemic heart disease. MethodsCIP4 conditional knock-out (CKO) mice were studied by echocardiography with strain analysis and histology following ischemia-reperfusion (I/R) injury and permanent left coronary artery (LCA) ligation to induce myocardial infarction. Wildtype C57BL/6NJ mice were transduced with adeno-associated virus (AAV) engineered for cardiomyocyte-specific expression of either a CaNA{beta}2 shRNA to inhibit CaNA{beta}2 expression, a VIVIT peptide to inhibit CaN-NFAT signaling, or a CaNA{beta}2 PP peptide to block CIP4-CaNA{beta}2 binding. AAV-transduced mice were studied by I/R injury. Additional mice were subjected to permanent LCA ligation and subsequently treated with AAV to test the effects of CaN inhibition in chronic ischemic cardiomyopathy. The effects of CaNA{beta}2 PP-GFP expression on primary T-cell activation were studied in vitro. ResultsCIP4 CKO mice and mice expressing the PP anchoring disruptor peptide exhibited preserved cardiac function after I/R injury and decreased infarct size and preserved cardiac function 8 weeks after myocardial infarction by permanent LCA ligation. In contrast, cardiomyocyte-specific depletion of CaNA{beta}2 and VIVIT peptide expression worsened outcome after I/R injury and in chronic ischemic cardiomyopathy. In addition, in contrast to cardiomyocytes, PP-mediated CaNA{beta} anchoring inhibition had no effect on T-cell activation and cytokine expression in vitro. ConclusionsCIP4-CaNA{beta}2 signalosomes promote adverse cardiac remodeling and are not cardioprotective. Proof-of-concept is provided for the treatment of ischemic cardiomyopathy by a PP anchoring disruptor gene therapy. Targeting these complexes may be beneficial in cardiovascular diseases, including ischemic cardiomyopathy and acute myocardial infarction. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LITargeting CIP4, which is a scaffold protein for the phosphatase calcineurin, improves cardiac function in mice after acute myocardial infarction due to ischemia-reperfusion injury and in chronic ischemic cardiomyopathy. C_LIO_LIGene therapy-based expression of a calcineurin A{beta}-derived polyproline peptide, which can compete CIP4-calcineurin binding, is beneficial in acute and chronic myocardial infarction. C_LI What Are the Clinical Implications?O_LIThis study establishes CIP4 signalosomes as a new drug target for the treatment of ischemia-reperfusion injury and chronic pathological cardiac remodeling. C_LIO_LIThis study provides proof-of-concept for a new gene therapy approach to treating acute myocardial infarction and chronic ischemic cardiomyopathy. C_LI

APALORD: An R-based tool for differential alternative polyadenylation analysis of long-read RNA-seq data

Zhang, Z. — 2025-06-17

Alternative polyadenylation (APA) is a critical co/post-transcriptional process that enhances RNA isoform diversity, regulating mRNA stability, localization and translation in a spatiotemporal manner. Over the past decade, long-read (LR) RNA sequencing techniques have advanced rapidly, producing datasets that could offer insights into APA mechanisms. Here we introduce APALORD (Alternative Polyadenylation Analysis of LOng-ReaDs), an R-based analysis tool for APA analysis of LR RNA-seq data. Leveraging precise 3 end information from 3-primed LR RNA-seq data, APALORD identifies polyadenylation sites (PASs) and quantifies PAS usage (PAU) at individual sites for each sample. It conducts APA analysis at the gene level (using Kolmogorov-Smirnov test) and at the level of individual PAS (using DEXSeq) across sample conditions. APALORD was applied to direct RNA-seq data from human embryonic stem cells (hESCs) and hESC-derived neurons. PASs were identified with high accuracy and a transcriptome-wide 3UTR lengthening trend was found, consistent with previous studies. APALORD analysis of PacBio cDNA data from human tissues confirmed a 3UTR lengthening trend in cortex compared to liver. R2C2 libraries generated on the Nanopore platform were analyzed with APALORD and it revealed APA change associated with polysome fractions in human neural progenitor cells. In summary, APALORD offers a comprehensive framework for differential APA analysis using LR RNA-Seq data, empowering researchers to investigate 3 end dynamics across diverse biological contexts.

Systems-level Consequences of Low RAF Abundance for EGFR-ERK Signaling

Lee, S. H. — 2025-06-17

The RAF kinases are central links between RAS, once activated by receptor tyrosine kinases (RTKs), and the extracellular signal-regulated kinases (ERK). In many cancer cells, RAFs are the least abundantly expressed RTK-ERK pathway proteins and can be present at just hundreds of copies per cell at the plasma membrane, but the consequences of limited RAF expression are unclear. By developing continuum and stochastic computational models of the epidermal growth factor receptor (EGFR)-ERK pathway, we showed that low RAF abundance creates stoichiometric bottlenecks between RTKs and ERK with concomitant stochastic RAF dynamics that propagate to weakly expressed downstream pathway proteins. Advanced sensitivity and Sloppiness analyses identified RAS activation and RAS-RAF interactions as strong determinants of signaling in low-RAF settings and revealed an efficient model fitting approach. RAF bottlenecks were predicted to impede ERK activation by oncogenic RAS mutants and explained a tendency for RAF1 membrane localization to be noisy. This work provides quantitative insight into a common, yet unexplored, regime for EGFR-ERK signaling and a systematic approach to develop and characterize dynamic models of receptor-mediated signaling. STATEMENT OF SIGNIFICANCERAF kinases connect receptors to the mitogenic ERK signaling pathway by translocating to the plasma membrane, but in a substantial fraction of cancer cell contexts RAFs are greatly outnumbered by other pathway proteins, potentially creating an unrecognized and consequential signaling bottleneck. We trained a novel computational model of EGFR-ERK signaling and characterized it comprehensively using integrated multivariate sensitivity analyses and Sloppiness analysis. The results revealed that low RAF abundance suppresses EGFR-mediated ERK activation, limits the effects of upstream oncogenic RAS mutants, and creates stochastic RAF dynamics that can propagate downstream. Thus, the canonical EGFR-ERK pathway exhibits divergent behaviors in a parameter space representative of a substantial fraction of cancer cell settings.

Lubricin's Mucin Domain Has Strong Polyproline Type-II Helical Character

Feng, B. N. — 2025-06-18

Lubricin is a glycoprotein that is crucial for maintaining joint health by preventing joint wear by reducing joint friction in the boundary mode. Lubricin was recently observed to hinder the formation of uric acid crystals in the joint and prevent a form of gouty arthritis. However, despite lubricins great physiological importance, our current understanding of the molecular origins of lubricins beneficial properties is limited by a lack of detailed structural information regarding its central mucin domain: lubricins large size (227.5 kDa) and numerous glycosylations pose a substantial obstacle to conventional experimental methods for solving protein structures. In this work, we employ a combination of physics-based replica exchange molecular dynamics (REMD) simulations and circular dichroism (CD) experiments to shed light on the structure of lubricins central mucin domain. Using REMD, we model [KEPAPTTP]2, an amino acid repeat found throughout the mucin domain, and find that the mucin domain is likely to exhibit polyproline type II (PPII) helices, which are further stabilized by O-linked oligosaccharide chains. Motivated by these simulation results, we performed circular dichroism spectroscopy on fragments of the mucin domain that also show clear polyproline-II helical character, corroborating our computational findings. Altogether, this work provides strong evidence of a lubricin mucin domain with significant polyproline type II content. As polyproline helices are often also found in other glycoproteins with antifreeze properties, this work may also explain the atomistic underpinnings of their interfacial functions, including lubrication and competition with crystal formation. SIGNIFICANCELubricin is a mucinous glycoprotein containing a central heavily glycosylated domain that plays a crucial role in the lubrication of joints. However, little is known about the structure of this large central mucin domain, which makes the development of related therapeutics or biomedical devices challenging. In this work, we provide strong evidence that lubricins mucin domain possesses polyproline type II (PPII) character that is enhanced by glycosylation upon the basis of a combination of molecular dynamics simulations and circular dichroism experiments. Lubricins PPII character provides a molecular basis for its lubricating properties, which may provide insights into related antifreeze glycoproteins (AFGP) and the development of new biocompatible lubricants and ryo-preservatives.

Stiffness sensing fuels matrix-driven metabolic reboot for kidney repair and regeneration

Gui, Y. — 2025-06-26

Kidney repair after acute kidney injury (AKI) relies on a finely tuned extracellular matrix (ECM) that provides structural integrity and mechanical cues. As primary ECM architects, fibroblasts and pericytes rapidly mobilize to the injury site post-AKI, yet the ECM-driven repair mechanisms remain incompletely defined. Here, leveraging tissue engineering, genetic and pharmacological models, and multi-omics, we profiled the proteome landscape of decellularized kidney matrix scaffold post-AKI and highlighted microfibrillar-associated protein 2 (Mfap2) as a key core matrisome component primarily sourced from fibroblasts and pericytes. Mfap2 loss disrupted kidney architecture and metabolism, aggravating AKI. Global proteomics revealed that Mfap2 deficiency suppressed tubular 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) expression via estrogen receptor 2 (Esr2)-mediated transcriptional repression and increased protein succinylation. Phosphoproteomics and spatial transcriptomics further demonstrated a shift in mechanical signaling, with Mfap2 loss hyperactivating mitogen-activated protein kinases and upregulating large tumor suppressor kinase 1 (Lats1) in tubular cells without altering integrin receptor activities. In turn, Lats1 suppressed Esr2 transcription independent of its canonical Yap/Taz effectors, without affecting ubiquitin-mediated Esr2 degradation. Therapeutically, Esr2 agonists restored kidney function in Mfap2-deficient models. These findings position Mfap2 as a key regulator of ECM dynamics and mechanosignaling, linking tissue stiffness to metabolic reprogramming required for kidney repair.

Transcriptomic Profiles from Normal and Tumor Tissue Samples Reveal Distinct Venule Populations and Novel Tumor Endothelial Cell Markers in Breast Cancer

Phoenix, K. N. — 2025-06-27

BackgroundThe breast tumor microenvironment (TME) is a complex milieu composed of many factors contributing to breast cancer (BC) heterogeneity and therapeutic resistance. Aberrant tumor vasculature in the TME limits nutrient and drug delivery, inhibits anti-tumor immunity, and contributes to a lack of cancer therapy efficacy. Utilizing publicly available scRNA-seq datasets, this study characterizes differences between normal breast and breast tumor endothelial cells (EC), provides insights into tumor endothelial cell subtypes, endothelial anergy, and identifies novel, tumor-specific vascular therapeutic targets. MethodsGene expression data from normal and breast tumor tissue samples were integrated, and the EC subset was extracted via canonical gene marker expression. The EC subset was clustered and evaluated for cell subtypes and differentially expressed genes (DEG). Normal EC (NEC) and tumor EC (TEC) markers were further assessed for correlation to bulk gene expression and patient survival outcomes in cBioPortal and Kaplan-Meier Plotter. Cell type gene expression specificity was evaluated in the 3CA single-cell RNA-seq datasets across multiple cancers. ResultsThis analysis revealed differences in NEC and TEC subtype populations. Breast NEC contained similar proportions of venule and capillary populations, while breast TEC demonstrated a majority of the venule subtype. Further, TEC venules were phenotypically distinct from the NEC venules. Consistent with endothelial anergy, suppression of the key adhesion protein SELE was noted, as well as several pro-inflammatory cytokines including IL6, CCL2, and CXCL8, likely downstream of aberrant NF-kB signaling. Differential gene expression analysis identified several TEC specific up-regulated genes compared to NEC, including CLEC14a, IGFBP4, EMCN, and ADM5. CLEC14a, EMCN, and ADM5 were further validated in the single-cell Curated Cancer Cell Atlas (3CA) to be highly specific to the endothelial cell clusters across multiple tumor types, while IGFBP4 was diversely expressed in endothelial, fibroblast, and some malignant cell types. ADM5, a novel tumor vascular marker, was enhanced in TEC venules and less so in arteriole or capillaries. High expression of ADM5 was associated with poor breast cancer patient survival in the basal PAM50 cancer subtype compared to normal and luminal subtypes. Further, across multiple cancer types, high ADM5 expression was associated with reduced patient survival in anti-PD1- and anti-CTLA4-treated patients but not in anti-PDL-treated patients. ConclusionsIntegration of single-cell RNA-seq data identified an anergic-like response in breast TEC and multiple, highly specific markers to TEC not found in normal breast tissue. CLEC14a and EMCN were validated as TEC markers, extending their annotation in breast TEC, and ADM5 identified as a novel TEC marker in breast and other cancers. Moreover, as ADM5 is associated with reduced patient overall survival, this data suggests that a better understanding of ADM5 and other TEC-specific response pathways may provide novel approaches to reactivate anergic TECs and lead to effective therapeutic interventions for cancer patients. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/661087v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@a81bf2org.highwire.dtl.DTLVardef@c2b983org.highwire.dtl.DTLVardef@216ab9org.highwire.dtl.DTLVardef@1e5bebb_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

CMV reshapes lymphoid immunity in aging: a single-cell atlas with predictive modeling

Grabauskas, T. — 2025-06-27

Cytomegalovirus (CMV) is a common herpesvirus that establishes lifelong latency and becomes increasingly prevalent with age. We systematically characterized CMV-associated immune remodeling by analyzing six human cohorts (two newly built) using single-cell RNA sequencing, T cell receptor (TCR) sequencing, and flow cytometry. Beyond the well-known expansion of CD4/CD8 TEMRA, adaptive NK, and {gamma}{delta} T cells, CMV(+) adults exhibited increased frequencies of GZMK CD8 T cells and atypical B cells, alongside a reduction of CD56dim NK cells. Longitudinal profiling of an individual who seroconverted revealed rapid CMV-driven shifts in circulating immune cell frequencies. Single-cell TCR data analyzed using a large database of CMV-associated clones combined with predictive modelling (CMVerify), identified novel CMV-specific clonal expansions reproduced across two independent cohorts. In the CD8 lineage, CMV-specific clones were enriched in GZMK CD8 and CD8 TEMRA cells, while in the CD4 lineage, Th1 cells showed clonal expansion alongside CD4 TEMRA cells. This integrative study revealed how latent CMV alters the cellular and clonal landscape, defining GZMK CD8 and Th1 cells as newly recognized elements of response to CMV in humans.

Targeting breast cancer senescence in 3D models of bone metastasis

Hamburger, E. C. B. — 2025-06-30

Chemotherapeutic treatment of breast cancer with Doxorubicin can induce tumor and stromal cell senescence leading to therapy-resistance. Senescence-associated secretory phenotype (SASP) promotes secretion of pro-inflammatory and tumorigenic factors causing systemic inflammation. Combined, this can result in immune suppression, tumor growth and secondary spread of cancer. Targeting and removing senescent and cancerous cells using a combination of chemotherapeutic and senolytic drugs may reduce systemic inflammation, improve therapeutic efficacy, and prevent metastasis. Treatment of both triple-negative breast cancer (MDA-MB-231) cells, and primary spine osteoblasts 0.25 {micro}M Doxorubicin showed significant induction of senescence indicated by p21 positive cells. Doxorubicin and senolytics (RG-7112, o-Vanillin) treatment of mono-culture and co-culture spheroids showed a significant additive effect on decreased tumor sphere viability and growth. This was correlated with decreased p21 and Ki67 proliferation marker in both the breast cancer and osteoblast cells. In all cases, combined Doxorubicin and senolytics significantly reduced sphere size and cancer cell outgrowth, indicating reduced metastatic potential. Future chemotherapeutic treatment of breast cancer patients may be optimized by adding senolytic drugs to more effectively clear tumors and help regenerate surrounding stroma tissue such as in the bone metastatic environment.

Segmental duplication-mediated rearrangements alter the landscape of mouse genomes

Francoeur, E. R. — 2025-07-22

Segmental duplications (SDs) are dynamic regions of mammalian genomes that drive structural variation and phenotypic diversity. However, genome-wide characterization of the SD landscape and the rearrangements mediated by SD paralogs in mouse genomes has been limited by the near-exclusive reliance on short-read sequencing technologies. Here, we integrate long-read genome assemblies, optical mapping, and k-mer based copy number analysis across eight genetically diverse inbred mouse strains to identify and characterize SD-mediated rearrangements. We identify 223 rearrangements affecting over 14 Mb of sequence and reveal substantial variation in gene content. These rearrangements affect loci involved in immunity, sensory perception, and gene regulation, including variation in the amylase gene cluster (Amy2a) and KRAB-zinc finger genes. We observed that SD flanks in mouse genomes are significantly enriched for young LINE-1 transposable elements, suggesting a potential role for transposons in promoting recombination and generating SDs within mice. Our findings highlight the contribution of SDs to genome structure and intra-species variation, and provide a resource for identifying regions prone to rearrangement in a critical model organism for biomedical research.

Profibrotic monocyte-derived alveolar macrophages as a biomarker and therapeutic target in systemic sclerosis-associated interstitial lung disease

Markov, N. S. — 2025-08-11

Interstitial lung disease (ILD) is present in over 60% of patients with systemic sclerosis (SSc) and is the leading cause of SSc-related deaths. Profibrotic monocyte-derived alveolar macrophages (MoAM) play a causal role in the pathogenesis of pulmonary fibrosis in animal models where their persistence in the niche requires signaling through Colony Stimulating Factor 1 Receptor (CSF1R). We hypothesized that the presence and proportion of MoAM in bronchoalveolar lavage (BAL) fluid from patients with SSc-ILD may be a biomarker of ILD severity. To test this hypothesis, we analyzed BAL fluid from 9 prospectively enrolled patients with SSc-ILD and 13 healthy controls using flow cytometry and single-cell RNA sequencing. Patients with SSc-ILD had more MoAM and interstitial macrophages in BAL fluid than healthy controls, and their abundance was associated with lung fibrosis severity. We identified changes in the MoAM transcriptome as a function of treatment with mycophenolate, an effective therapy for SSc-ILD. In SSc-ILD lung explants, spatial transcriptomics identified an expanded population of interstitial macrophages spilling into the alveolar space. Our findings suggest that the proportion of profibrotic MoAM and interstitial macrophages in BAL fluid may serve as a biomarker of SSc-ILD and credential them as possible targets for therapy.

Paired plus-minus sequencing is an ultra-high throughput and accurate method for dual strand sequencing of DNA molecules

Cheng, A. P. — 2025-08-14

Distinguishing real biological variation in the form of single-nucleotide variants (SNVs) from errors is a major challenge for genome sequencing technologies. This is particularly true in settings where SNVs are at low frequency such as cancer detection through liquid biopsy, or human somatic mosaicism. State-of-the-art molecular denoising approaches for DNA sequencing rely on duplex sequencing, where both strands of a single DNA molecule are sequenced to discern true variants from errors arising from single stranded DNA damage. However, such duplex approaches typically require massive over-sequencing to overcome low capture rates of duplex molecules. To address these challenges, we introduce paired plus-minus sequencing (ppmSeq) technology, in which both DNA strands are partitioned and clonally amplified on sequencing beads through emulsion PCR. In this reaction, both strands of a double-stranded DNA molecule contribute to a single sequencing read, allowing for a duplex yield that scales linearly with sequencing coverage across a wide range of inputs (1.8-98 ng). We benchmarked ppmSeq against current duplex sequencing technologies, demonstrating superior duplex recovery with ppmSeq, with a rate of 44%{+/-}5.5% (compared to [~]5-11% for leading duplex technologies). Using both genomic as well as cell-free DNA, we established error rates for ppmSeq, which had residual SNV detection error rates as low as 7.98x10-8 for gDNA (using an end-repair protocol with dideoxy nucleotides) and 3.5x10-7{+/-}7.5x10-8 for cell-free DNA. To test the capabilities of ppmSeq for error-corrected whole-genome sequencing (WGS) for clinical application, we assessed circulating tumor DNA (ctDNA) detection for disease monitoring in cancer patients. We demonstrated that ppmSeq enables powerful tumor-informed ctDNA detection at concentrations of 10-4 across most cancers, parts per million sensitivity in cancers with high mutation burden, and further increased sensitivity with higher sequencing depth. We then leveraged genome-wide trinucleotide mutation patterns characteristic of urothelial (APOBEC3-related and platinum exposure-related signatures) and lung (tobacco-exposure-related signatures) cancers to perform tumor-naive ctDNA detection, showing that ppmSeq can identify a disease-specific signal in plasma cell-free DNA without a matched tumor, and that this signal correlates with imaging-based disease metrics. Altogether, ppmSeq provides an error-corrected, cost-efficient and scalable approach for high-fidelity WGS that can be harnessed for challenging clinical applications and emerging frontiers in human somatic genetics where high accuracy is required for mutation identification.

Dose-dependent interferon programs in myeloid cells after mRNA and adenovirus COVID-19 vaccination

Eryilmaz, G. N. — 2025-08-18

The SARS-CoV-2 pandemic provided a rare opportunity to study how human immune responses develop to a novel viral antigen delivered through different vaccine platforms. However, to date, no study has directly compared immune responses to all three FDA-approved COVID-19 vaccines at single-cell multi-omic resolution. We longitudinally profiled SARS-CoV-2-naive adults (n=31) vaccinated with BNT162b2, mRNA-1273, or Ad26.COV2.S, integrating plasma cytokines, antibody titers, and single-cell multi-omic data (DOGMA-seq). We discovered a distinct, transient interferon program (ISG-dim) that emerged specifically 1-2 days after the first mRNA dose in [~]10% of myeloid cells. This state was characterized by ISGF3 complex activation and its target genes (e.g., MX1, MX2, DDX58), with transcriptional and epigenetic profiles distinct from the robust interferon program observed after mRNA boosting or a single Ad26.COV2.S dose (ISG-high). In vitro stimulation of human monocytes showed that IFN- alone recapitulates ISG-dim, whereas both IFN- and IFN-{gamma} are required for ISG-high. These findings define dose-dependent interferon programming in human myeloid cells, highlight mechanistic differences between priming and boosting, with implications for optimizing vaccine platform choice, dose scheduling, and formulation.

High-Resolution Spatial Transcriptomics Reveals Fibroblast and Neuroimmune Microenvironments in Endometriosis Lesions

Haney, C. M. — 2025-08-25

Endometriosis is a chronic, systemic, inflammatory disease characterized by the presence of endometrium-like tissue growing outside of the uterus. One of its main symptoms is chronic pain and inflammation leading to a decreased quality of life. This is a common disease, as at least one in ten female-born individuals have endometriosis. Yet the understanding of the mechanisms that drive pain symptoms and disease progression remain poorly defined. This study establishes the precise spatial transcriptomic cartography of human ovarian and peritoneal lesions, two of the most commonly found lesions. We identified shared spatial features across lesion types, including immune cell infiltration, fibroblast specific compartments surrounding epithelial glands, and distinct distributions of neuronal and macrophage subsets. We precisely defined sensory neuronal subtypes, and mapped their spatial location relative to immune cells. We further validate the epithelial-neuronal interactome, using an in vitro 3D model of peripheral sensory brain organoids co-cultured with human endometriosis epithelial and fibroblast cells. By mapping spatial cellular interactions and identifying conserved features across lesion types linked to pain, our study provides emerging insights into endometriosis pathophysiology, paving the way for the development of novel targeted therapeutic strategies.

Biomolecular phase boundaries are described by a solubility product that accounts for variable stoichiometry and soluble oligomers

Akram, S. A. — 2025-08-29

The solubility product is a rigorous description of the phase boundary for salt precipitation and has previously been shown to qualitatively describe the condensation of biomolecules. Here we present a derivation of the solubility product showing that the solubility product is also a robust description of biomolecule phase boundaries if care is taken to account for soluble oligomers and variable composition within the dense phase. Our calculation describes equilibrium between unbound monomers, the dense phase, and an ensemble of oligomer complexes with significant finite-size contributions to their free energy. The biomolecule phase boundary very nearly resembles the power law predicted by the solubility product when plotted as a function of the monomer concentrations. However, this simple form is concealed by the presence of oligomers in the dilute phase. Accounting for the oligomer ensemble introduces complexities to the power law phase boundary including re-entrant behavior and large shifts for stoichiometrically matched molecules. We show that allowing variable stoichiometry in the dense phase expands the two phase region, which appears as curvature of the phase boundary on a double-logarithmic plot. Furthermore, this curvature can be used to predict variations in the dense phase composition at different points along the phase boundary. Finally, we show how the solubility product power law can be identified in experiments by using dilute phase dissociation constants to account for the oligomer ensemble.

Senolytic-Resistant Senescent Cells Have a Distinct SASP Profile and Functional Impact: The Path to Developing Senosensitizers

Tripathi, U. — 2025-08-30

The senescent cell (SC) fate is linked to aging, multiple disorders and diseases, and physical dysfunction. Senolytics, agents that selectively eliminate 30-70% of SCs, act by transiently disabling the senescent cell anti-apoptotic pathways (SCAPs), which defend those SCs that are pro-apoptotic and pro-inflammatory from their own senescence-associated secretory phenotype (SASP). Consistent with this, a JAK/STAT inhibitor, Ruxolitinib, which attenuates the pro-inflammatory SASP of senescent human preadipocytes, caused them to become "senolytic-resistant". Administering senolytics to obese mice selectively decreased abundance of the subset of SCs that is pro-inflammatory. In cell cultures, the 30-70% of human senescent preadipocytes or human umbilical vein endothelial cells (HUVECs) that are senolytic-resistant (to Dasatinib or Quercetin, respectively) had increased p16INK4a, p21CIP1, senescence-associated {beta}-galactosidase (SA{beta}gal), {gamma}H2AX, and proliferative arrest similarly to the total SC population (comprising senolytic-sensitive plus -resistant SCs). However, the SASP of senolytic-resistant SCs entailed less pro-inflammatory/ apoptotic factor production, induced less inflammation in non-senescent cells, and was equivalent or richer in growth/ fibrotic factors. Senolytic-resistant SCs released less mitochondrial DNA (mtDNA) and more highly expressed the anti-inflammatory immune evasion signal, glycoprotein non-melanoma-B (GPNMB). Transplanting senolytic-resistant SCs intraperitoneally into younger mice caused less physical dysfunction than transplanting the total SC population. Because Ruxolitinib attenuates SC release of pro-apoptotic SASP factors, while pathogen-associated molecular pattern factors (PAMPs) can amplify the release of these factors rapidly (acting as "senosensitizers"), senolytic-resistant and senolytic-sensitive SCs appear to be interconvertible.

Comparison of a long-read amplicon sequencing approach to short-read amplicons for microbiome analysis

O'Sullivan, B. — 2025-09-05

Most microbiome studies to date rely on sequencing short amplicons of the 16S rRNA gene on Illuminas platforms. Because of the short read length, sequences often can be identified reliably only to the family or genus levels. Long read sequencing with whole-length 16S rRNA sequencing can improve taxonomic resolution, but often only to the species level. StrainID is an alternative approach that amplifies a large segment of the ribosomal operon, including the entire 16S rRNA gene, internal transcribed spacer, and a portion of the 23S rRNA gene. This longer amplicon is designed to allow ribotype-level classification. Although studies have demonstrated the utility of StrainID for several sample types, it has not yet been validated for saliva. Here, we compared the performance of StrainID to short read amplicons with saliva samples as well as a synthetic mock DNA community and human and mouse fecal samples. Short reads were amplified with primer pairs appropriate for the corresponding sample type, and were classified with two different taxonomic databases. For both saliva and fecal samples, we found that StrainID performed similarly to short reads overall and demonstrated a key benefit with phylogenetic-based beta diversity tests and taxonomic classification. Our results further build on establishing StrainID as a valid method and specifically validate its use with saliva samples. ImportanceThe interpretation of microbiome composition studies is highly dependent on the methodologies chosen during experimental design, which affects factors such as resolution, throughput, cost, and accuracy. StrainID is an approach that can improve resolution while maintaining high-throughput and similar costs to short-read sequencing. The salivary microbiome represents a diverse community of microbes with links to a variety of health conditions and disease states. Closely related strains of bacteria can have drastically different effects on their host. Establishing StrainID as a valid approach for studying the salivary microbiome opens avenues for research that improve upon alternative methods by increasing sensitivity and accuracy compared to traditional short read approaches.

Redefined Strategies to generate Conditional miR-141/200c miRNA cluster Knockout mice to eliminate confounding neo cassettes

Verma, R. — 2025-09-07

MicroRNAs (miRNAs) of the miR-200 family specifically miR-141 and miR-200c regulate neurogenesis, differentiation, and epithelial-mesenchymal transitions in development and several diseases including cancer and stroke. The STOCK Mirc13tm1Mtm /Mmjax mouse line, which targets the miR-141/200c cluster, was originally generated and described by Park et al. 2012 as a conditional "knockout-first" allele requiring a two-step breeding strategy: FLP recombination to excise lacZ/neo cassettes followed by Cre recombination to delete the floxed miRNA cluster (1). However, subsequent studies either bypassed this step and reported knockouts based on direct crosses with Cre mouse lines, leaving residual lacZ/neo sequences that may silence upstream elements or introduce transcriptional artifacts or rare studies used less efficient FLPe Deleter mice. Here, we present a detailed and refined strategy to conditional miR-141/200c knockouts mice using FLPo Deleter mice to efficiently eliminate lacZ/neo cassettes. Our approach not only confirmed complete deletion of miR-141 and miR-200c in various organs such olfactory bulbs and lungs where these miRNAs are robustly expressed using various approach such as genotyping qPCR validation and in situ hybridization but showed that without the use of FLPo deleter mice deletion of miR-141/200c cluster amy also lead to loss of several close proximity physiologically important genes such as ptpn6, phb2, atn1 and eno1. By restoring a clean floxed allele using FLPo deleter mice prior to Cre deletion, we establish a reliable and interpretable mouse model for dissecting the roles of the miR-141/200c cluster miRNA in various disease models.

REV1 inhibition enhances trinucleotide repeat mutagenesis

Siegel, A. — 2025-09-16

Trinucleotide repeat (TNR) instability has been implicated in the pathogenesis of numerous neurodegenerative disorders. Because TNR instability causes mutagenesis of the underlying gene, we refer to the repeat instability phenomenon as TNR mutagenesis in this study. While germline expansions destabilize TNR to cause disease anticipation, somatic cell TNR instability drives earlier onset of symptoms and further disease progression. However, the drivers behind these repeat length changes remain unclear. Current models suggest that DNA replication slippage events and the action of genome instability pathways, such as DNA repair, cause TNR mutagenesis. Whether mutagenic polymerases from the translesion synthesis (TLS) pathway result in TNR instability is unclear. TLS polymerases are best at bypassing difficult-to-replicate DNA regions due to bulky lesions or gaps in DNA. While some effects of TLS polymerases on TNR instability have been explored in lower organisms, evidence in human cells is lacking. Using a quantitative GFP reporter with expanded CAG repeats, we show that inhibition of the TLS polymerase REV1 by its inhibitor, JH-RE-06, or siRNA knockdown increases TNR instability and the underlying mutability. These results suggest that REV1 protects Trinucleotide repeat length mutagenesis through potential continuous DNA synthesis when replicative polymerases stall ahead of repeat secondary structures. Collectively, we present evidence of the role of the TLS pathway in TNR instability, with potential implications for understanding mutability mechanisms, disease biology, and therapeutic targeting.

Brinker regulates reciprocal outcomes of BMP signal between stem cells and differentiating cells

Poursaeid, S. — 2025-09-16

Drosophila male germline stem cells (GSCs) reside at the testis tip, surrounding a cluster of niche cells known as the hub. Bone Morphogenetic Protein (BMP) ligands secreted from the hub exert both contact-dependent and -independent effects. In close proximity to the niche, BMP signaling maintains stem cells by suppressing transcription of the key differentiation factor Bag of Marbles (Bam). In contrast, the diffusible fraction of BMP promotes differentiation of cells by activating bam. How a single signaling pathway produces such opposing outcomes has remained unclear. Here, we show that the diffusible BMP fraction induces bam transcription by repressing the transcriptional repressor Brinker (Brk). We further found that brk mRNA displays a highly heterogeneous expression pattern within interconnected spermatogonia, suggesting that Brk may prime cell fate in a subset of transit-amplifying cells, helping to preserve a population poised for dedifferentiation while maintaining other cells for differentiation. Our findings propose a model in which a single niche-derived factor modulates reciprocal outcomes inside versus outside the niche, which is essential for the tissue homeostasis. Given the broad use of BMP signaling across stem cell niches, this mechanism may represent a general strategy to ensure correct balance between self-renewal and differentiation of stem cells.

Spoti-find: A novel, open-source void spot assay image analysis tool

Hardy, C. C. — 2025-10-01

The void spot assay (VSA) is a widely used, non-invasive method for evaluating urinary behavior in rodents, but existing analysis tools are limited in scope, throughput, or accessibility. We developed Spoti-find, a stand-alone, open-source VSA image analysis application that introduces novel, biologically meaningful metrics including void circularity, distance to paper edge, and volume-based binning into primary, micro-, and nanovoids. Designed with usability in mind, Spoti-find features a graphical interface that enables manual or semi-automated spot identification, adjustable thresholds, and streamlined data export without the need for coding expertise. We validated Spoti-find across diverse datasets, showing strong inter-user consistency, sensitivity to known phenotypes in aging and disease models, and accuracy in capturing novel parameters while demonstrating high agreement with existing tools. By capturing behavioral context and spatial morphology in voiding patterns, Spoti-find expands the interpretive power of VSA and provides a flexible, user-friendly platform for phenotyping urinary dysfunction in preclinical studies.

spCorr: flexible and scalable inference of spatially varying correlation in spatial transcriptomics

Jiang, C. — 2025-10-02

Spatial transcriptomics has transformed our ability to explore gene expression within its tissue context, enabling us to dissect subtle yet biologically significant variations in situ. While numerous computational methods have been proposed for detecting Spatially Varying Genes (SVGs) expression by modeling each individual gene separately, much less effort has been devoted to understanding how correlations between genes change across space. Such Spatially Varying Correlations (SVCs) are critical for understanding biological processes such as gene regulatory mechanisms shaped by local tissue environments, yet existing tools remain limited for this task. To address this gap, we present spCorr, a flexible and scalable regression framework for studying SVCs. spCorr provides interpretable, spot-level estimates of gene correlation and detects gene pairs whose correlations vary across locations or between tissue domains. Through extensive simulations and real-data analyses, we show that spCorr achieves high detection power, reliably controls the False Discovery Rate (FDR), and is computationally efficient. Importantly, spCorr reveals biologically meaningful correlation patterns that highlight fine-scale tissue structures, gene module functions, and region-specific interactions, offering new opportunities to study coordinated gene regulation in spatial transcriptomics.

Cyp7b1-inhibiting azoles as novel enhancers of hematopoietic stem and progenitor cell mobilization

Vu, B. L. — 2025-10-14

Mobilized hematopoietic stem and progenitor cells (HSPCs) are essential for transplantation-based therapies, including curative gene therapies for sickle cell disease (SCD). While granulocyte colony-stimulating factor (G-CSF, filgrastim) remains the standard mobilization agent, many patients respond inadequately, and it can trigger life-threatening vaso-occlusive crises in SCD. The CXCR4 antagonist AMD3100 (plerixafor) is routinely combined with G-CSF for non-SCD settings but is ineffective as a single agent in SCD, underscoring the urgent need for alternative strategies. We previously identified 27-hydroxycholesterol (27HC) as a physiological inducer of HSPC mobilization during pregnancy. Here, we show that exogenous 27HC enhances AMD3100-induced HSPC mobilization in mice, either alone or with G-CSF. Because 27HC is metabolized by the enzyme Cyp7b1, we tested whether pharmacological Cyp7b1 inhibition could mimic this effect. Treatment with clotrimazole, an antifungal and Cyp7b1 inhibitor, significantly enhanced AMD3100-induced HSPC mobilization in wild-type, SCD, and humanized mice. Importantly, intravenous administration of voriconazole, a clinically approved systemic antifungal with Cyp7b1-binding activity, similarly augmented AMD3100-induced HSPC mobilization in wild-type and SCD mice without altering steady-state hematopoiesis. These findings establish Cyp7b1-inhibiting azoles as novel and clinically relevant enhancers of HSPC mobilization, particularly for SCD patients who cannot safely receive G-CSF but require robust HSPC yields for gene therapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/682145v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1fddaaforg.highwire.dtl.DTLVardef@12036f4org.highwire.dtl.DTLVardef@1f8010aorg.highwire.dtl.DTLVardef@10c4aff_HPS_FORMAT_FIGEXP M_FIG C_FIG

Genetic interaction of Specc1l and Thm1 reveals cytoskeletal - ciliary crosstalk

Hufft-Martinez, B. M. — 2025-11-05

Cilium formation and actin cytoskeleton dynamics are interconnected, with evidence showing that elevated filamentous actin (F-actin) negatively regulates primary cilia length. Loss of the cytoskeletal protein SPECC1L, which itself does not localize to cilia, leads to increased F-actin and shortened cilia. Depolymerizing F-actin in Specc1l mutant cells restored cilia lengths, substantiating this inverse relationship. In cells harboring a Specc1l allele lacking only the coiled-coil domain 2, intracellular regions with both elevated and reduced F-actin were observed together with cilia shortening. Notably, F-actin was decreased at the ciliary base, suggesting that a different F-actin subpopulation contributes to the inverse relationship. We also identified a genetic interaction between Specc1l and Thm1, which encodes an intraflagellar transport-A (IFT-A) protein. Double or compound heterozygotes for Specc1l and Thm1 exhibited a higher penetrance of cleft palate compared to Specc1l heterozygotes alone. Together, these findings reveal a role for SPECC1L in cytoskeletal regulation of ciliogenesis affecting palate development.

SF3B1 phosphorylation is an evolutionarily conserved step in spliceosome activation carried out by the divergent, OTS964-insensitive kinase CRK9 in trypanosomes

Machida, K. — 2025-11-21

SF3B1 is a subunit of the heptameric SF3B complex which, as part of the U2 small nuclear ribonucleoprotein, facilitates branch point recognition in pre-mRNA splicing. In addition to this early-stage function, it was recently shown that activation of the spliceosome depends on the phosphorylation of threonine-proline (TP) motifs in SF3B1s N-terminal domain (NTD) by cyclin-dependent kinase 11 (CDK11). This breakthrough result was made possible by the discovery of the CDK11-specific inhibitor OTS964. Trypanosomes are protistan parasites whose proteomes are highly divergent in sequence from those of model organisms, and thus their CDKs were generically named CDC2-related kinases (CRKs). We previously characterized the trimeric CRK9 complex of Trypanosoma brucei and showed that it is essential for spliced leader trans splicing, the predominant splicing mode in the parasite. Although CRK9 and CDK11 deviate from each other substantially, we show that CRK9 activity is required to maintain SF3B1 phosphorylation in vivo, CRK9 directly phosphorylates TP motifs in the SF3B1 NTD in vitro, and the TP motifs themselves are crucial for spliceosome activation, demonstrating evolutionary conservation of this essential splicing step. Contrary to CDK11 and human cells, CRK9 and trypanosomes were rather insensitive to OTS964, indicating potentially exploitable differences in their ATP-binding pockets.

Encoding without generation in the medial prefrontal cortex during cautious goal-directed actions

Sajid, M. S. — 2025-11-28

Adaptive behavior under threat requires deciding when to act and when to withhold action to avoid harm, often under conditions where movement, arousal, and task demand covary. Medial prefrontal cortex (mPFC) activity is widely associated with such control, yet it remains unclear whether this activity reflects causal action generation or broader evaluative processes shaped by behavioral state. Here, we combined fiber photometry, single-cell calcium imaging, mixed-effects modeling, and optogenetic inhibition to examine how GABAergic neurons in mouse mPFC represent cues, actions, and outcomes during a series of learned avoidance tasks of increasing complexity that promote cautious responding. By explicitly controlling for baseline activity and movement, we show that much apparent task-related activity in mPFC reflects movement and cue-evoked signals that are also present in a control cortical region, the visual cortex. mPFC GABAergic neurons showed little encoding of simple avoidance contingencies but broadly encoded punished outcomes. A small subset of neurons with strong movement sensitivity encoded more demanding avoidance contingencies requiring selection between action generation and deferment. For equivalent avoidance actions, distinct neuronal populations preferentially encoded either cue onset or the action. Despite this encoding, optogenetic inhibition of mPFC had minimal effects on the learning or performance of the different contingencies. These findings reveal a dissociation between neural encoding and causal necessity, indicating that mPFC GABAergic activity primarily reflects evaluative and contextual aspects of cautious avoidance behavior rather than direct control of action execution. Significance statementAvoiding danger often requires deciding when to act and when to hold back. The dorsomedial prefrontal cortex (mPFC) is widely assumed to support this control, yet the contributions of its neurons have remained unclear. Using neural population and single-neuron recordings together with targeted inhibition in mice performing learned threat-guided tasks, we show that GABAergic mPFC neurons are activated by cues, actions, and outcomes, but are not required for executing the behavior itself. These findings suggest that the mPFC primarily evaluates and contextualizes threat-motivated actions rather than generating them, highlighting a common mismatch between neural encoding and causal necessity. Synopsis for ReviewersNote the following organizational features: O_LIGiven the hierarchical structure of the experiments, all analyses use linear mixed-effects models, with sessions nested within mice as random effects. Models include relevant covariates, such as movement and baseline activity, to disentangle their contributions from task contingency-related effects. C_LIO_LITo facilitate reading flow, statistical details supporting the Results are reported in the figure legends. Additional statistical values can be provided or relocated to the main text if preferred. C_LIO_LIFigures and their legends are placed adjacent to the corresponding results to improve readability, but high-resolution versions are also at the end. In all population plots, the symbols, and traces are Mean{+/-}SEM. If error bars are not visible, they are smaller than the symbol and trace. We can adjust this as requested. C_LIO_LISeveral analyses are presented as supplemental figures to streamline the main narrative; figure order and presentation can be adjusted as requested. C_LI The Results are organized into six segments: O_LImPFC and visual cortex GABAergic neurons are sensitive to movement. Using fiber photometry, we show that GABAergic neurons in both mPFC and a control cortical region, visual cortex (VI), exhibit strong sensitivity to movement (Fig. 1; Fig. 1-S1). These findings motivated the inclusion of movement and baseline activity as covariates in neural analyses to control their effects. C_LIO_LIBehavioral performance across a series of avoidance tasks. Mice were trained in tasks of increasing difficulty (outlined in Fig. 2A). Animals were first exposed to three neutral cues (noUS), followed by cues predicting different contingencies. In AA19, CS1 signals active avoidance, and then in AA39, CS2 signals passive avoidance. Behavioral performance is shown in Fig. 2, associated movement traces in Fig. 2-S1, and mixed-effects models of movement dynamics in Fig. 2-S2. C_LIO_LIFiber photometry reveals limited encoding of simple avoidance but encoding of aversive outcomes. Fiber photometry recordings from mPFC and VI during the tasks show that mPFC GABAergic neurons do not robustly encode simple avoidance contingencies but exhibit sensitivity to more complex task demands and punished errors. Analyses also tested neural responses to unsignaled aversive stimulation. Results are shown in Fig. 3, with photometry traces in Fig. 3-S1 and a full model spanning all task phases in Fig. 3-S2. C_LIO_LISingle-cell recordings reveal movement-sensitive classes with distinct contingency encoding. Miniscope imaging was performed in mPFC during AA19 and AA39. Neurons were classified based on their correlation with movement and then tested for contingency encoding. This revealed class-specific differences, including a subset of neurons that encode avoidance-related variables. Results for AA19 are shown in Fig. 4, and for AA39 in Fig. 4-S1. C_LIO_LIAvoidance actions segregate into distinct movement modes with dissociable neural encoding. Avoidance actions were classified based on their movement profiles, revealing three response modes that differ in response latency and vigor, reflecting varying degrees of behavioral caution. Clustering neural activity within each mode identified neurons that selectively encode cue onset versus action execution (Fig. 5 and Fig. 5-S1). C_LIO_LIOptogenetic inhibition of mPFC has minimal effects on avoidance behavior. Using two complementary optogenetic approaches (eArch3.0 and Vgat-ChR2), we tested the causal contribution of mPFC neurons to avoidance behavior. Inhibition produced minimal effects on learning or performance across tasks. Behavioral effects and optogenetic validation are shown in Fig. 6. C_LI

Trans-regulation of heterochromatin underlies genetic variation in 3D genome contacts

Fortin, H. J. — 2025-12-12

Genetic variation drives phenotypic diversity and disease susceptibility. Trans-acting genetic variation coordinates genome-wide chromatin changes, yet the molecular mechanisms underlying this regulation remain unclear. Here, we use the power of mouse genetics to investigate how genetic variation at trans-acting loci regulates 3D chromatin interactions. Using HiChIP to map H3K27ac-associated regulatory elements in C57BL/6J and DBA/2J embryonic stem cells (ESCs), we identified 4,962 strain-differential interactions, 71% of which overlapped chromatin accessibility quantitative trait loci (QTL), establishing chromatin interaction variation is predominantly heritable. These differential interactions showed coordinated changes in chromatin state and gene expression, with stronger interactions associated with increased accessibility and transcription. Notably, loci regulated in trans exhibited a unique chromatin signature where weaker interactions were enriched for H3K9me3-marked heterochromatin. Analysis of F1 hybrids revealed dominant repressive effects, consistent with heterochromatin-mediated trans-regulation. To causally test this mechanism, we generated reciprocal congenic mouse strains carrying a Chr13 trans-QTL region. Integrated multiomic profiling of congenic ESC lines demonstrated that this single locus coordinates changes in H3K9me3, H3K27ac, chromatin accessibility, and 3D contact frequency at hundreds of distal targets. Consistent with our expectations, 73-83% of differential interactions at Chr13 trans-QTL targets changed in the predicted direction, demonstrating that heterochromatin-mediated trans-regulation coordinately regulates hundreds of regulatory loci. This work establishes heterochromatin formation as a mechanism by which genetic variation at trans-acting loci coordinates changes across chromatin accessibility, histone modifications, and 3D genome organization, providing a framework for understanding how early developmental chromatin states could generate phenotypic variation while preserving essential developmental programs.

Mechanisms of HSV-1 helicase-primase inhibition and replication fork complex assembly

Yu, Z. — 2025-12-24

Herpesviruses are widespread double-stranded DNA viruses that establish lifelong latency and cause various diseases. Although DNA polymerase-targeting antivirals are effective, increasing drug resistance underscores the need for alternatives. Helicase-primase inhibitors (HPIs) are promising antivirals, but their mechanisms of action are poorly defined. Furthermore, how the helicase-primase (H/P) complex and DNA polymerase coordinate genome replication is not well understood for herpesviruses. Here, we report cryo-EM structures of the herpes simplex virus (HSV) H/P complex bound to HPIs, showing that these lock the helicase-primase complex in an inactive conformation. Single-molecule assays reveal that HPIs cause helicase-primase complexes to pause in unwinding activity on DNA. The structure of an HPI-bound replication fork complex, comprising the H/P complex (UL5, UL52, and UL8) and polymerase holoenzyme (UL30 and UL42), reveals a previously uncharacterized interface bridging these complexes. These findings provide a structural framework for understanding herpesvirus replisome assembly and advancing inhibitor development.

A scaffold attachment factor PHM-2 regulates synaptic transmission through SLO-2 potassium channel in C. elegans

Niu, L. — 2025-12-26

Scaffold attachment factor B (SAFB) proteins are evolutionarily conserved DNA/RNA binding proteins that are involved in multiple processes of gene expression. These proteins are broadly expressed with particular high expression observed in the nervous system. However, their physiological roles in neurons are largely unclear. Here we show that PHM-2, the sole SAFB ortholog in C. elegans, regulates synaptic transmission at the neuromuscular junctions through an effect on SLO-2 potassium channel. We found that phm-2 knockout suppresses a sluggish phenotype of worms expressing a hyperactive SLO-2 channel, greatly reduces SLO-2-mediated neuronal whole-cell currents, and enhances neuromuscular synaptic transmission. In addition, we found that PHM-2 genetically interacts with another DNA/RNA binding protein, HRPU-2/hnRNP U, to control SLO-2 expression through a posttranscriptional mechanism. These results reveal a novel function of a SAFB protein in regulating neuronal activity, and may help understand the physiological roles of SAFB proteins in the nervous system of other species. Author SummaryProteins in the SAFB family are found in many species, and they help control how genes are expressed in cells. These proteins are commonly present in the nervous system, but their exact roles in nerve cells are not well understood. In this study, we examined the single SAFB-like protein called PHM-2 in the nematode C. elegans to learn how it affects the nervous system. We discovered that PHM-2 plays an important role in communication between nerve cells and muscles. Worms lacking PHM-2 were able to counteract the sluggish movement caused by a hyperactive potassium channel called SLO-2. Without PHM-2, nerve cells had much smaller electrical currents mediated by SLO-2 and a stronger signaling from nerves to muscles. We also found that PHM-2 works together with another genetic regulator, HRPU-2, to control the amount of SLO-2 protein made in nerve cells. These findings reveal a new role for SAFB proteins in shaping neuronal activity by regulating potassium channels. Understanding this process in worms may provide clues about how these proteins contribute to brain function in humans.

Subthalamic nucleus controls cautious action timing under threat

Zhou, J. — 2025-12-30

Adaptive goal-directed actions performed under the threat of punishment introduce a demand for caution, often expressed as delayed response timing that balances urgency against error risk by allowing more time for cognitive evaluation. Although this form of temporal regulation is essential for survival, its underlying neural mechanisms remain poorly understood. We show that glutamatergic neurons in the subthalamic nucleus (STN) regulate the timing of cued actions to avoid harm. Optogenetic activation of the STN, or its projections to the midbrain but not the globus pallidus, modulates action timing in a frequency-dependent manner, accelerating initiation to the extent that animals can no longer respond cautiously, defer actions, or stop initiated actions. These results show that the STN shapes goal-directed behavior by gating action-initiation timing, establishing its midbrain projections as a key circuit for balancing urgency and caution under threat. Significance statementThis work shows that activity in the subthalamic nucleus (STN) helps set the timing of actions triggered by warning cues. By activating STN neurons and their projections to the midbrain, we found that stimulation made animals respond faster, and high-frequency activation eliminated their ability to delay actions when caution was required or to withhold actions that would otherwise be punished. These findings highlight a key brain pathway that governs when actions are initiated, a function that is often disrupted in neurological and psychiatric disorders.

GerAB Residues Predicted to Interact with Water Based on MD Simulations Mediate Germinosome Stability in Bacillus subtilis spores

Chen, L. — 2026-01-10

Some species in the Bacillales and Clostridiales orders form spores under unfavourable environmental conditions. These spores are metabolically dormant and highly resistant to extreme stress. The spore core--analogous to the protoplast of vegetative cells--contains only 25-45% water by wet weight, compared to ~80% in vegetative cells. Upon activation by small-molecule nutrients, spores germinate, restoring their core water content, restoring metabolism and becoming easy to kill, while progressing through outgrowth to vegetative growth. GerAB is the B subunit of the prototypical Bacillus subtilis GerA GR (germinant receptor), a membrane protein belonging to the Amino Acid--Polyamine-Organocation (APC) superfamily of transporters. It functions as the L-alanine sensor that initiates germination and was previously predicted, based on molecular dynamics (MD) simulations, to contain a putative water channel. Using MD simulations, we identified low amount of water permeating through GerAB (ranging from 1-121 water molecule/{micro}s in 10 parallel MD simulations), thus revealing a water pathway in GerAB that diverges from the L-alanine binding pocket, suggesting that water transport may play roles in germination beyond facilitating ligand binding. Analysis of water-residue contact frequencies identified eight hydrophilic residues lining this path. Individual substitution of high-contact residues with similarly sized non-polar residues impaired L-alanine germination and disrupted GerAB structural integrity as assessed by Western Blotting. These mutants also respond to the AGFK germinant mixture (L-asparagine, D-glucose, D-fructose and potassium) in slower, yet individually distinct kinetics compared to that of wt spores. These findings prove that water contact residues in GerAB predicted by MD simulations are crucial for the stability of this protein and thus the germinosome complex with all GRs.

Conserved assembly architecture of the essential herpesvirus packaging accessory factor

Bailey, E. J. — 2026-01-22

To create a new wave of infectious virions, all herpesviruses require an accessory factor of unknown function to package their viral genomes into nascent capsids. Here, we present cryo-EM structures of the packaging accessory factor from the -herpesvirus herpes simplex virus type 1 (HSV-1, UL32) and the {beta}-herpesvirus human cytomegalovirus (HCMV, UL52). Unlike homologs from the {gamma}-herpesviruses, neither UL32 nor UL52 form stable homopentameric rings. UL52 forms incomplete pentameric rings lacking one or two protomers. UL32 does not form stable higher-order species, but stabilization through chemical crosslinking revealed a novel quaternary structure where three pentameric rings assemble into a "tripentamer." Our results reveal that herpesvirus packaging accessory factors adopt distinct oligomeric states but are constrained to pentameric symmetry. Assembly of protomers into a ring creates a positively charged central channel that we show is critical for infectious virus production in HSV-1. Taken together, our study points to a structurally conserved, essential function of packaging accessory factors across the Herpesviridae.

CuiT is a Cu importer required for metal homeostasis in Salmonella enterica

Zhao, Z. — 2026-01-23

Copper (Cu) is an essential micronutrient that serves as a cofactor for redox enzymes but becomes toxic when unregulated. In bacteria, while Cu efflux systems are well characterized, mechanisms of Cu import remain poorly understood. Here, we characterize the major facilitator superfamily transporter CuiT (STM1486) as a key Cu importer in Salmonella enterica. Comparative genomics revealed that cuiT is evolutionarily conserved across Enterobacteriaceae, and structural modeling predicts a 12-transmembrane-helix architecture with conserved His, Met, and Cys residues suitable for Cu coordination. Functional analyses demonstrated that deletion of cuiT reduces intracellular Cu accumulation, slows Cu uptake kinetics, and diminishes expression of Cu-responsive genes, including copA, cueP, cueO, and golB. Conversely, overexpression of CuiT increases intracellular Cu but sensitizes cells to Cu stress, highlighting the need for tight regulation. Kinetic modeling indicates that CuiT mediates rapid Cu import, supporting larger intracellular Cu pools compared to Pseudomonas influx transporters. These findings position CuiT as a central component of the Salmonella Cu homeostasis network, linking Cu import to transcriptional regulation, redox balance, and stress adaptation. Our work provides mechanistic insights into bacterial Cu acquisition and suggests CuiT and associated pathways as potential targets for antimicrobial strategies. SignificanceCopper (Cu) is essential for bacterial redox enzymes but toxic when dysregulated. While Cu efflux pathways are well studied, mechanisms of Cu import are poorly understood. We identify CuiT, a conserved major facilitator superfamily transporter, as a key Cu importer in Salmonella enterica. CuiT controls intracellular Cu levels, influences Cu-responsive gene expression, and maintains redox balance and stress adaptation. Disruption or overexpression of CuiT perturbs Cu homeostasis, highlighting its regulatory importance. These findings reveal a critical bacterial Cu acquisition pathway and suggest CuiT and its network as potential antimicrobial targets, advancing understanding of metal homeostasis in pathogens.

CMTM6-Silencing Microbial Immunotherapy Reprograms PDAC Tumors and Restores T-cell Function

Chabu, C. Y. — 2026-01-28

Despite recent advances in immunotherapy for advanced malignancies, Pancreatic ductal adenocarcinoma (PDAC) remains largely refractory to current immunotherapy due to dense fibrosis, limited antigen presentation, and myeloid-driven immune suppression. Here we report the tumor-targeting, immune remodeling, and safety profiles of the attenuated Salmonella enterica serovar Typhimurium strain CRC2631, and of iSTORM, a next-generation derivative engineered for tumor-localized CMTM6 silencing. CRC2631 preferentially colonizes orthotopic and genetically engineered PDAC tumors, with enrichment in primary lesions and metastases. Tumor-localized CRC2631 induces chemokine and adhesion programs consistent with leukocyte recruitment, increases intratumoral activated T-cell fractions, and triggers transcriptional signatures aligned with innate sensing, interferon signaling, antigen-processing and presentation, and apoptosis programs. iSTORM extends this platform by delivering CMTM6-targeting shRNA to modulate a PD-L1-stabilizing, myeloid-associated immune-evasion programs within tumor-colonized tissue. Compared with CRC2631, iSTORM increases intratumoral CD8+ T cells, shifts T-cell state toward activation with reduced exhaustion-prone features, strengthens antigen-presentation programs, and achieves deeper tumor control. A lyophilized formulation preserves immune remodeling while improving deployability. Mechanistically, glycan arrays and functional studies support mannose-rich glycan-guided tumor engagement. iSTORM toxicity studies, including systemic cytokine, hematologic, blood chemistry, and lethality demonstrate a favorable safety profile. Collectively, these findings establish iSTORM as a safe, programmable, CMTM6-silencing microbial immunotherapy platform that selectively targets and penetrate PDAC tumors to unleash anti-tumor immune activities. What is already known on this topicPDAC is highly resistant to immune checkpoint blockade because dense stroma and myeloid-dominated suppression prevent effective T-cell infiltration; attenuated Salmonella strains can selectively colonize tumors but first-generation agents showed limited efficacy and safety concerns. What this study addsThis study defines CRC2631/iSTORM as a tumor-selective microbial immunotherapy that exploits surface-exposed, mannose-rich N-glycans to colonize PDAC, delivers CMTM6 silencing, and restores CD8+ T-cell activation and tumor control in models resistant to PD-1 blockade immunotherapy. How this study might affect research, practice or policyThese findings provide a mechanistic blueprint for glycan-guided, CMTM6-targeted bacterial "living drugs," support rational combination strategies for deepening therapeutic effect, and establish a lyophilized, biocontained platform that could be developed into scalable microbial immunotherapies for PDAC and other immunologically cold solid tumors.

Glycolytic Specialization Shapes Neuronal Physiology and Function in vivo

Wolfe, A. D. — 2026-02-18

Neurons perform diverse functions that impose distinct energetic demands, but how energy-metabolic pathways are matched to these functions in vivo remains unknown. Here we show that two functionally divergent sister chemosensory neurons in C. elegans, ASEL and ASER, exhibit asymmetric glycolytic flux, with ASER exhibiting high and ASEL having low levels of glycolysis. Metabolic imaging, metabolic network modeling, and electrophysiology measurements reveal that ASERs elevated glycolysis supports a hyperpolarized resting potential, low input resistance, and rapid repolarization that enable a distinct functional role compared to ASEL. Impairing glycolysis collapses these electrophysiological specializations without abolishing neuronal excitability, and selectively disrupts ASERs calcium responses while leaving ASEL largely unaffected. These findings demonstrate that neuron-specific glycolytic programs shape core biophysical properties and are required for functional identity in vivo, establishing metabolism as an active determinant of neuronal physiology.

PXL: a Nucleic Acid-Binding Module of Promyelocytic Leukemia Protein

Fairchild, D. — 2026-02-20

The promyelocytic leukemia protein (PML) is a stress-response factor that assembles into PML nuclear bodies, dynamic subnuclear compartments involved in tumor suppression and antiviral defense. The most abundant isoform, PML-1, has been linked to transcriptional regulation, genome stability, and antiviral responses, yet the molecular basis of these functions remains unclear. Here, we report that PML-1 contains a unique nucleic acid- binding module, PXL, and determine its three-dimensional structure by X-ray crystallography. Further biochemical, mutational, and cellular analyses, including RNA-seq, demonstrate that this module selectively binds single-stranded G-rich RNA and DNA motifs and modulates the transcriptome. These findings reveal an unexpected molecular function of PML and provide a framework for understanding its roles in nuclear organization and gene regulation.

A RAD18 SAP domain PIP motif enables PCNA mono-ubiquitination and USP1-BRCA1 synthetic lethality

Ashton, N. W. — 2026-02-27

The proliferating cell nuclear antigen (PCNA) sliding clamp is a central component of eukaryotic DNA synthesis. In response to DNA damage, PCNA is mono-ubiquitinated by the RAD6 (E2)-RAD18 (E3) complex. However, the structural basis by which RAD18 engages PCNA to direct mono-ubiquitination has remained poorly defined. Mono-ubiquitinated PCNA can subsequently be extended with K48-linked poly-ubiquitin chains that target PCNA for degradation. Ubiquitin-specific protease 1 (USP1) reverses both mono- and poly-ubiquitination of PCNA; accordingly, USP1 inhibition promotes accumulation of mono-ubiquitinated PCNA at replication forks and reduces total PCNA levels, leading to replication defects that are synthetically lethal with BRCA1 deficiency. Here, we combine computational and structural approaches to identify and characterize a SAP domain PCNA-interacting peptide (PIP) motif within RAD18. We demonstrate that this interaction is required for DNA damage-induced PCNA mono-ubiquitination and for PCNA turnover following USP1 loss. Disruption of the RAD18-PCNA interface suppresses ssDNA gap accumulation and reduces USP1 inhibitor sensitivity in BRCA1-deficient cells. Furthermore, cells adapted to prolonged USP1 inhibition exhibit reduced RAD18 levels, suggesting that deregulation of PCNA mono-ubiquitination represents a biologically relevant resistance mechanism. Together, these findings define a structural interface required for RAD18-dependent PCNA mono-ubiquitination and establish it as a key determinant of USP1-BRCA1 synthetic lethality. Significance StatementE3 ubiquitin ligases act as substrate-specificity factors, yet the structural mechanisms by which they direct ubiquitination by partner E2 enzymes remain poorly defined. Here, we identify a critical RAD18-PCNA interaction interface required for efficient PCNA mono-ubiquitination and turnover in USP1-deficient cells. USP1 inhibition is synthetically lethal with BRCA1 loss and USP1 inhibitors are entering clinical development for BRCA1-deficient breast and ovarian cancers, making it essential to define determinants of response and resistance. Disruption of the RAD18-PCNA interface suppresses USP1 inhibitor-induced replication defects and cytotoxicity, while cells adapted to prolonged inhibition exhibit reduced RAD18 levels. These findings establish RAD18-dependent PCNA regulation as a mechanistic determinant of response to USP1-targeted therapy.

High-pH NMR to Identify Macromolecular Hydrogen-Bonds and Foldons

Alexandrescu, A. — 2026-03-03

Hydrogen bond (H-bond) restraints are critical for NMR structure determination, yet their experimental identification can be challenging for marginally stable structures that afford insufficient protection from (H/D) exchange in D2O. As an alternative, we explored the use of NMR between pH 10 and 11 conditions that promote rapid exchange, for identifying backbone amide protons involved in H-bonds. We analyzed [~]750 amide sites distributed across ten proteins with known structures. Persistence of amide protons at high pH in standard 2D 1H-15N HSQC spectra for 15N-labeled proteins in H2O, or TOCSY for unlabeled proteins, identifies H-bonds with [~]91% accuracy that exceeds the [~]80% accuracy of traditional H/D exchange experiments in D2O. For two -helical coiled coils and three globular proteins, we performed alkaline unfolding experiments taking advantage of amide NMR signal attenuation from unstructured polypeptides. Increasing the sample pH led to a progressive loss of native amide proton NMR signals, revealing an unfolding hierarchy where "foldons" remaining at the highest pH values had the most persistent H-bonds under EX1 exchange conditions. The foldons observed at high pH are consistent with partially folded structures previously characterized near neutral pH by native state hydrogen exchange, equilibrium unfolding, and protein fragment studies. For {beta}-sheet proteins, foldons correspond to regions with high inter-residue contact density, whereas in coiled coils they demarcate regions with high -helical propensity. High-pH NMR experiments provide a sensitive, fast, inexpensive, and broadly applicable approach to map H-bonding in marginally stable or partially folded proteins. Additionally, they offer the opportunity to explore uncharted protein dynamics and unfolding pathways under basic pH conditions.

Brain-wide mapping and synaptic localization of C1QL3 using a novel epitope-tagged knock-in mouse

Armstrong, W. — 2026-03-09

Synapse formation and function are coordinated spatially and temporally by a host of synaptic proteins that regulate neuronal signaling, synapse specificity, and plasticity; many of which are implicated in neuropsychiatric disorders. Members of the C1q/TNF superfamily function as synaptic organizers, shaping synapse assembly and maintenance. Among them, C1QL3 plays a putative role in trans-synaptic adhesion and modulation of synaptic strength, but the lack of a reliable antibody to detect it has severely limited the ability to map its endogenous localization and study its biochemical properties. Here, we present a novel epitope-tagged knock-in mouse line (C1ql32HA), in which two hemagglutinin (HA) epitopes were inserted near the N-terminus of the endogenous C1QL3 protein. This model enables purification, detection, and subcellular localization of native C1QL3 protein (C1QL3-2HA) with high specificity, eliminating the need for overexpression or custom antibodies. We validated that C1ql32HA mice maintain normal mRNA expression, biochemical properties, and behavior. Using native PAGE, we determined the endogenous oligomeric state of C1QL3-2HA. Brain-wide light-sheet microscopy uncovered an expanded neuroanatomical map of C1QL3-2HA expression, including newly identified populations in cortical and subcortical regions as well as the retina. Dual immunohistochemistry confirmed cell type-specific expression patterns, and super-resolution STED microscopy localized C1QL3-2HA to hippocampal mossy fiber synapses, positioned between pre- and post-synaptic markers, supporting its hypothesized role in trans-synaptic complexes. This knock-in mouse line is a powerful tool for studying the anatomical, molecular, and synaptic biology of C1QL3 in all cellular/tissue contexts, enabling future studies into its potential roles in the nervous system and beyond.

MEX3B is a positive pan-inflammasome regulator

Cahoon, J. G. — 2026-04-01

Inflammasomes lead to activation of inflammatory caspases, which induce pyroptosis and an inflammatory immune response to control microbial infections. Inflammasomes are tightly regulated to avoid lethal sepsis and chronic autoimmune conditions. However, posttranslational regulation of inflammatory caspases remains poorly defined. We constructed 375 individual ubiquitin ligase knockout lines by CRISPR-Cas9, performed an unbiased screening, and identified Muscle Excess 3B (MEX3B), an RNA-binding protein and ubiquitin ligase, as a positive regulator of the caspase-4 inflammasome. Genetic depletion of MEX3B inhibited not only the caspase-4 but also NLRP3 and NLRC4 inflammasomes, regarding caspase activation, pyroptosis, and secretion of inflammasome-dependent cytokines, in human cells and murine primary macrophages. This MEX3B function required its RNA-binding, but not ubiquitin ligase activity. These results suggest that MEX3B is a pan-inflammasome regulator and a potential therapeutic target for inflammation.