bioRxiv Channel: University of Guelph

Coexistence in Periodic Environments

Scott, A. M. — 2023-02-27

Climate change and other anthropogenic impacts are rapidly altering natural environmental periodicities on a variety of time scales. Despite this, a general theoretical foundation describing the role of periodic environmental variation in structuring species interactions and ecological communities is still underdeveloped. Alarmingly, this leaves us unprepared to understand and predict implications for the maintenance of biodiversity under global change. Here, we extend a two-species Lotka-Volterra competition model that incorporates periodic forcing between seasons of high and low production to investigate the effects of changing environmental patterns on species coexistence. Towards this, we define coexistence criteria for periodic environments by approximating isocline solutions akin to classical coexistence outcomes. This analytical approach illustrates that periodic environments (i.e., seasonality) in and of themselves can mediate different competitive outcomes, and these patterns are general across varying time scales. Importantly, species coexistence may be incredibly sensitive to changes in these abiotic periods, suggesting that climate change has the potential to drastically impact the maintenance of biodiversity in the future.

Specialized cingulo-opercular network activation in judging ambiguity of facial expressions in a slow reveal fMRI paradigm

Pierce, J. E. — 2023-02-27

Cortical task control networks, including the cingulo-opercular (CO) network play a key role in decision-making across a variety of functional domains. In particular, the CO network functions in a performance reporting capacity that supports successful task performance, especially in response to errors and ambiguity. In two studies testing the contribution of the CO network to ambiguity processing, we presented a valence bias task in which masked clearly and ambiguously valenced emotional expressions were slowly revealed over several seconds. This slow reveal task design provides a window into the decision-making mechanisms as they unfold over the course of a trial. In the main study, the slow reveal task was administered to 32 young adults in the fMRI environment and BOLD time courses were extracted from regions of interest in three control networks. In a follow-up study, the task was administered to a larger, online sample (n = 81) using a more extended slow reveal design with additional unmasking frames. Positive judgments of surprised faces were uniquely accompanied by slower response times and strong, late activation in the CO network. These results support the initial negativity hypothesis, which posits that the default response to ambiguity is negative and positive judgments are associated with a more effortful controlled process, and additionally suggests that this controlled process is mediated by the CO network. Moreover, ambiguous trials were characterized by a second CO response at the end of the trial, firmly placing CO function late in the decision-making process.

Species Delimitation Under Allopatry: Genomic Divergences Within and Across Continents in Lepidoptera

Joshi, M. — 2023-03-07

Delimitation of allopatric populations into species remains subjective and largely arbitrary. Many cold-adapted species from the subarctic and Central and Southern European Mountain systems display frequent allopatry with disjunct distributions of their populations. The same concerns Holarctic species which are many and which almost regularly show various degrees of differentiation between the continents. In this study, we analyze high- throughput target enrichment data for ten groups of arctic-alpine and Holarctic lepidopteran species sampled from four main regions across the Holarctic realm - Fennoscandia, North America, Alps and Altai. We first aimed to assess whether the genetic differences in the nuclear genome reflect observed DNA barcode divergences and second, whether the gap between population and species-level differences can be dissected using genomic data. We compared the phylogenetic trees and uncorrected pairwise genetic distances obtained from target enrichment and the mitochondrial cytochrome oxidase I (COI) barcodes for each of the study species. Additionally, we also performed a suite of population genetic and species delimitation analyses to further shed light on patterns of intraspecific variation using a large number of nuclear markers. We observed that in about one half of the cases, DNA barcodes tended to show phylogenetic relationships similar to the target enrichment markers. We report varying levels of nuclear genetic differentiation among the populations analyzed, starting from low differentiation of geographically separated populations to the deeper separation of some Nearctic population and further arctic-alpine disjunction. Given that no single consistent pattern emerged across different case studies, we demonstrate that the delimitation of allopatric populations into species could be done much more efficiently and in a consistent manner if based on a large set of universal genetic loci, which would help in reaching standards for taxonomic delimitation of allopatric populations.

Comparative analysis of syngeneic mouse models of high-grade serous ovarian cancer

Cook, D. P. — 2023-03-10

Ovarian cancers often exhibit high rates of recurrence and poor treatment response. Preclinical models that recapitulate the heterogeneity of human disease are critical to develop new therapeutic approaches. While patient-derived models are a powerful tool for testing various therapeutics, their dependence on immune-compromised mice is severely limiting. Syngeneic mouse models, however, allow for the generation of tumours comprising the full repertoire of non-malignant cell types. Here we have performed a comparative analysis of diverse models of high-grade serous ovarian cancer based on transcriptomic profiling of 22 cell line models, and intrabursal and intraperitoneal tumours from 12 models. Among cell lines, we identify distinct features in signalling activity, such as elevated inflammatory signalling in STOSE and OVE16 models, and MAPK/ERK signalling in ID8 and OVE4 models; metabolic features, such as predicted reduction in glycolysis associated with subsets of engineered ID8 subclones; and relevant functional properties, including differences in EMT activation, PD-L1 and MHC class I expression, and predicted chemosensitivity. Finally, we evaluate variability in properties of the tumour microenvironment among models. We anticipate that this work will serve as a valuable resource, providing new insight to help in the selection of models for specific experimental objectives.

Unravelling bird nest arthropod community structure using metabarcoding

Levesque-Beaudin, V. — 2023-03-12

Bird nests are fascinating microcosms harboring a wide range of arthropods parasitizing the nesting birds or feeding on prey remains, feces, and the nest material. Studies of these communities have been entirely based on emergence traps which collect live organisms out of the nests. The analysis of nest contents and environmental DNA (eDNA) via metabarcoding could expand our knowledge and identify prey, exuviae, and other animal remains in bird nests. Here, we investigated the potential of arthropod remains, nest dust, and feathers to better describe taxonomic diversity accumulated in 20 bird nests collected in Guelph (Canada). We used subsampling strategies and tested two extraction approaches to investigate the distribution of DNA in nests, account for low-quality DNA, and the presence of inhibitory substances. In total, 103 taxa were detected via metabarcoding. Arthropod remains delivered the highest number of taxa (n=67), followed by nest dust (n=29). Extractions with the PowerSoil kit outperformed DNeasy extractions coupled with PowerClean Pro inhibitor removal. The subsamples of the same nest showed 5.5% and 47.1% taxonomic overlap for arthropod remains and PowerSoil extracted nest dust, respectively, indicating a heterogeneous eDNA distribution in nests. Most detected species were either feeding in the nest, i.e., herbivorous / predatory, or bird food. We also detected molecular traces of 25 bird species, whose feathers were likely used as nest material. Consequently, the metabarcoding of bird nest materials provides a more complete picture of nest communities, which can enable future studies on functional diversity and better comparisons between nesting species.

Arabidopsis calmodulin-like proteins CML13 and CML14 interact with proteins that have IQ domains

Teresinski, H. J. — 2023-03-12

In response to Ca2+ signals, the evolutionarily-conserved Ca2+ sensor calmodulin (CaM) regulates protein targets via direct interaction. Plants possess many CaM-like (CML) proteins, but their binding partners and functions are mostly unknown. Here, using Arabidopsis CML13 as bait in a yeast two-hybrid screen, we isolated putative targets from three, unrelated protein families, namely, IQD proteins, calmodulin-binding transcriptional activators (CAMTAs), and myosins, all of which possess tandem isoleucine-glutamine (IQ) structural domains. Using the split-luciferase complementation assay in planta and the yeast 2-hybrid system, CML13 and CML14 showed a preference for interaction with tandem over single IQ domains. Relative to CaM, CML13 and CML14 displayed weaker signals when tested with the non-IQ, CaM-binding domain of glutamate decarboxylase or the single IQ domains of CNGC20 (cyclic-nucleotide gated channel-20) or IQM1 (IQ motif protein1). We examined IQD14 as a representative tandem IQ-protein and found that only CaM, CML13, and CML14 interacted with IQD14 among 12 CaM/CMLs tested. CaM, CML13, and CML14 bound in vitro to IQD14 in the presence or absence of Ca2+. Binding affinities were in the nM range and were higher when two tandem IQ domains from IQD14 were present. Green fluorescent protein-tagged versions of CaM, CML13, and CML14 localized to both the cytosol and nucleus in plant cells but were partially relocalized to the microtubules when co-expressed with IQD14 tagged with mCherry. These and other data are discussed in the context of possible roles for these CMLs in gene regulation via CAMTAs and cytoskeletal activity via myosins and IQD proteins.

Predominant myosin super-relaxed state in canine myocardium with naturally occurring dilated cardiomyopathy

Ochala, J. — 2023-03-12

Dilated cardiomyopathy (DCM) is a naturally occurring heart failure condition in humans and dogs, notably characterized by a reduced contractility and ejection fraction. As the identification of its underlying cellular and molecular mechanisms remain incomplete, the aim of the present study was to assess whether the molecular motor myosin and its known relaxed conformational states are altered in DCM. For that, we dissected and skinned thin cardiac strips from left ventricle obtained from six DCM Doberman Pinschers and six non-failing controls (NF). We then used a combination of Mant-ATP chase experiments and X-ray diffraction to assess both energetic and structural changes of myosin. Using the Mant-ATP chase protocol, we observed that in DCM dogs, the amount of myosin molecules in the ATP-conserving conformational state also known as super-relaxed (SRX), is significantly increased when compared with NF dogs. This alteration can be rescued by applying EMD-57033, a small molecule activating myosin. Conversely, with X-ray diffraction, we found that in DCM dogs, there is a higher proportion of myosin heads in the vicinity of actin when compared with NF dogs (1,0 to 1,1 intensity ratio). Hence, we observed an uncoupling between energetic (Mant-ATP chase) and structural (X-ray diffraction) data. Taken together, these results may indicate that in the heart of Doberman Pinschers with DCM, myosin molecules are potentially stuck in a non-sequestered but ATP-conserving SRX state, that can be counterbalanced by EMD-57033 demonstrating the potential for a myosin-centered pharmacological treatment of DCM. New & noteworthyThe key finding of the present study is that, in left ventricles of dogs with a naturally occurring dilated cardiomyopathy, relaxed myosin molecules favor a non-sequestered super-relaxed state potentially impairing sarcomeric contractility. This alteration is rescuable by applying a small molecule activating myosin known as EMD-57033.

CanFlyet: Habitat Zone and Diet Trait Dataset for Diptera Species of Canada and Greenland

Majoros, S. E. — 2023-03-16

BackgroundTrue flies (Diptera) are an ecologically important group that play a role in agriculture, public health and ecosystem functioning. As researchers continue to investigate this order, it is beneficial to link the growing occurrence data to biological traits. However, large-scale ecological trait data are not readily available for fly species. While some databases and datasets include fly data, many ecologically relevant traits for taxa of interest are not included. In this study we create a dataset containing ecological traits (habitat and diet) for fly species of Canada and Greenland having occurrence records on the Barcode of Life Data Systems (BOLD). We present a dataset containing trait information for 981 Diptera species. New InformationDiptera were chosen for the dataset based on the occurrence records available for Diptera species from Canada and Greenland on the Barcode of Life Data Systems (BOLD). Trait data were then compiled based on literature searches conducted from April 2021 - April 2024 and assigned at the lowest taxonomic level possible. Three biological traits were included: habitat, larval diet, and adult diet. The dataset contains traits for 981 species across 380 genera, 34 subfamilies, and 61 families. This dataset allows for assignment of traits to occurrence data for Diptera species and can be used for further research into the ecology, evolution, and conservation of this order.

Geographic variation in evolutionary rescue in a predator-prey system under climate change: an example with aphids and ladybird beetles

Ge, X. — 2023-03-24

Under climate change, species can adapt to changing environments through phenotypic plasticity and natural selection, and this kind of evolutionary adaptation can vary geographically. Most species distribution models (SDMs) are built upon the "Niche conservatism" assumption. They often ignore the possibility of "evolutionary rescue" and underestimate species future range limits under climate change. Here, we select aphids and ladybirds as model species and develop an eco-evolutionary model to explore evolutionary rescue in a predator-prey system under climate change. We model the adaptive change of species thermal performance, accounting for biotic interactions of unique life-history trait. Our results show that there is geographic variation in evolutionary rescue for ladybirds (the predator) across different locations in the United States, with ladybirds being more likely to be rescued from extinction in southeastern locations. The possibility of rescue is primarily influenced by the change in seasonality. Our findings also indicate the additive genetic variance of predators has a stronger influence on the phenotype evolution and population dynamics of both prey and predators, compared to the additive genetic variance of the prey. Our research emphasizes the importance of incorporating evolutionary adaptation when predicting species range shift under climate change. The eco-evolutionary model framework can be applied to study the effect of evolution on interacting species population abundance and geographic distribution under climate change.

Integration of homeostatic and adaptive oxidative responses by a putative co-chaperone, Wos2, drives fungal virulence in cryptococcosis

Ball, B. — 2023-04-03

The increasing prevalence of invasive fungal pathogens are dramatically changing the clinical landscape of infectious diseases and are an imminent burden to public health that lack the resources (i.e., robust antifungals) to tackle this threat. Specifically, the human opportunistic pathogen, Cryptococcus neoformans, expresses elaborate virulence mechanisms and is equipped with sophisticated adaptation strategies to survive in harsh host environments. In this study, we extensively characterize Wos2, an Hsp90 co-chaperone homologue, featuring bilateral functioning for both cryptococcal adaptation and virulence strategies. Here, we evaluated the proteome and secretome signatures of Wos2 in enriched and infection-mimicking conditions to reveal a Wos2-dependent regulation of oxidative stress response. The wos2{Delta} strain reports defective intracellular and extracellular antioxidant protection systems measurable through a decreased abundance of critical antioxidant enzymes and reduced growth in the presence of peroxide stress. Additional Wos2-associated stress phenotypes were observed upon fungal challenge with heat shock, osmotic, and cell wall stressors. We demonstrate the importance of Wos2 for C. neoformans intracellular lifestyle during in vitro macrophage infection and provide evidence for wos2{Delta} reduced phagosomal replication levels. Accordingly, wos2{Delta} featured significantly reduced virulence in a murine model of cryptococcosis. Our study highlights a vulnerable point in the fungal chaperone network that offers a powerful druggable opportunity to interfere with both virulence and fitness. Author SummaryThe global impact of fungal pathogens, both emerging and emerged, is undeniable and the alarming increase in antifungal resistance rates hampers our ability to protect the global population from deadly infections. For cryptococcal infections, a limited arsenal of antifungals and resistance demands alternative therapeutic strategies, including an anti-virulence approach, which disarms the pathogen of critical virulence factors, empowering the host to remove the pathogen and clear the infection. To this end, we apply state-of-the-art mass spectrometry-based proteomics to interrogate the impact of a recently defined novel co-chaperone, Wos2, towards cryptococcal virulence using in vitro and in vivo models of infection. We defined global proteome and secretome remodeling driven by the protein and uncovered a novel role in modulating the fungal oxidative stress response. Complementation of the proteome findings with in vitro infectivity assays demonstrated a protective role for Wos2 within the macrophage phagosome, influencing fungal replication and survival. These results underscore differential cryptococcal survivability and weakened patterns of dissemination in the absence of wos2. Overall, our study establishes Wos2 as an important contributor to fungal pathogenesis and warrants further research into critical proteins within global stress response networks as potential druggable targets to reduce fungal virulence and clear the infection.

Effects of VCD-induced ovarian failure on single muscle fiber contractility in a mouse model of menopause

Mashouri, P. — 2023-04-05

ObjectiveMenopause is associated with impairments in muscle contractile function. The temporal and mechanistic basis of this dysfunction are not known. Using a mouse model of menopause we identified how gradual ovarian failure affects single muscle fiber contractility. Study designMice were injected with VCD over 15 days and ovarian failure developed over 120 days. Mice were then sacrificed and slow-type soleus (SOL) and fast-type extensor digitorum longus (EDL) muscles were dissected and chemically permeabilized for mechanical testing. Main outcome measuresMuscle fiber contractility was assessed via: force, rate of force redevelopment, instantaneous stiffness, and calcium sensitivity across three relative force levels (pCa10,pCa50,pCa90). ResultsPeak force and cross-sectional area (CSA) of the SOL were [~]33% and [~]24% greater in the VCD group as compared with controls (P<0.05), respectively, with no differences in force produced by the EDL fibers across groups (P>0.05). Upon normalizing force to CSA there were no differences across groups (P>0.05). Rate of force development was [~]33% faster for SOL in the VCD group compared to control. Ca2+ sensitivity did not differ between groups for either muscle at pCa50 (P>0.05). In the VCD group, Ca2+ sensitivity was higher for EDL, but lower for SOL at pCa10 and pCa90 (P<0.05), respectively. ConclusionsIn our mouse model of menopause, alterations to muscle contractility were much less evident as compared with ovariectomized models. This divergence across models highlights the importance of better approximating the natural trajectory of menopause during and after the transitional phase of ovarian failure on neuromuscular function.

Resolving the temporal splenic proteome during fungal infection for discovery of putative dual perspective biomarker signatures

Muselius, B. — 2023-04-08

Fungal pathogens are emerging threats to global health with the rise of incidence associated with climate change and increased geographical distribution; factors also influencing host susceptibility to infection. Accurate detection and diagnosis of fungal infections is paramount to offer rapid and effective therapeutic options. For improved diagnostics, the discovery and development of protein biomarkers presents a promising avenue; however, this approach requires a priori knowledge of infection hallmarks. To uncover putative novel biomarkers of disease, profiling of the host immune response and pathogen virulence factor production is indispensable. In this study, we use mass spectrometry-based proteomics to resolve the temporal proteome of Cryptococcus neoformans infection of the spleen following a murine model of infection. Dual perspective proteome profiling defines global remodeling of the host over a time course of infection, confirming activation of immune associated proteins in response to fungal invasion. Conversely, pathogen proteomes detect well-characterized C. neoformans virulence determinants, along with novel mapped patterns of pathogenesis during the progression of disease. Together, our innovative systematic approach confirms immune protection against fungal pathogens and explores the discovery of putative biomarker signatures from complementary biological systems to monitor the presence and progression of cryptococcal disease.

Prefrontal layer V pyramidal neurons comprise multiple subtypes with distinct nicotinic responses and projection targets

Patel, A. V. — 2023-04-11

The neurotransmitter acetylcholine supports goal-directed cognitive functions via activation of its nicotinic and muscarinic classes of receptors within the prefrontal cortex. These receptors are expressed on pyramidal neurons located within layer V of the prefrontal cortex, which integrate afferent signals and contribute toward cognitive circuits via efferent projections to cortical and subcortical targets. Using whole-cell electrophysiology, retrograde labelling, and neuron reconstruction in the juvenile mouse prefrontal cortex, we identified three unique isoform-specific nicotinic receptor responses that are present in distinct subtypes of layer V pyramidal neurons. Broadly, we observed 7 or 7/{beta}2* nicotinic responses in burst-firing neurons that project to the contralateral cortex or nucleus accumbens, respectively, and {beta}2* nicotinic responses in regular-firing neurons that project to the ventromedial thalamus. These findings provide insight into a receptor isoform-specific mechanism by which nicotinic acetylcholine neurotransmission may support cognitive functions via modulation of distinct efferent projections from this brain region.

ENOD93 interacts with cytochrome c oxidase altering respiratory ATP production and root growth in plants

Lee, C. P. — 2023-04-16

The early nodulin 93 (ENOD93) gene family in plants can regulate biological nitrogen fixation in legumes and nitrogen use efficiency in cereals but its molecular function is unknown. We show profile hidden Markov models define ENOD93 as a distant homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 is reported to regulate cytochrome oxidase (CIV) influencing the generation of a mitochondria proton motive force in yeast. Knockout of enod93 in Arabidopsis leads to a short root phenotype. ENOD93 is associated with a protein complex the size of CIV in isolated mitochondria but neither CIV abundance nor its activity in ruptured organelles changed in enod93. However, a progressive loss of ADP-dependent respiration rate was observed in enod93 mitochondria which could be fully recovered in complemented lines. Mitochondrial membrane potential was higher in enod93 but ATP synthesis and ADP depletion rates progressively decreased. Respiration rate of whole enod93 seedlings was elevated and root ADP content was nearly double that in WT without a change in ATP content. These altered energetic states correlated with elevated respiratory substrate levels in roots of enod93 compared to WT and complemented lines. Overexpression of ENOD93 lowered ATP content in roots and increased the abundance of a range of amino acids in both roots and leaves. We propose that two previously unconnected gene families in plants, ENOD93 and HYPOXIA INDUCED GENE DOMAIN, are the functional equivalent of yeast RCF2 but have remained undiscovered in many eukaryotic lineages because they are encoded in two separate genes. Highlight significanceWe identify the enigmatic early nodulin ENOD93 gene family as the plant homolog of the N-terminal regulatory domain of the yeast RESPIRATORY SUPERCOMPLEX 2 (RCF2) of the mitochondrial oxidative phosphorylation system and provide biochemical and physiological evidence of its role in plant ATP production, broadly explaining the role of ENOD93 in plants.

Expression of E. coli glycogen branching enzyme in an Arabidopsis mutant devoid of endogenous starch branching enzymes induces the synthesis of starch-like polyglucans

Laura Boyer — 2015-05-29

Starch synthesis requires several enzymatic activities including branching enzymes (BEs) responsible for the formation of (1[->]6) linkages. Distribution and number of these linkages are further controlled by debranching enzymes (DBEs) that cleave some of them, rendering the polyglucan water-insoluble and semi-crystalline. Although the activity of BEs and DBEs is mandatory to sustain normal starch synthesis, the relative importance of each in the establishment of the plant storage polyglucan (i.e. water-insolubility, crystallinity, presence of amylose) is still debated. Here, we have substituted the activity of BEs in Arabidopsis with that of the Escherichia coli glycogen branching enzyme (GlgB). The latter is the BE counterpart in the metabolism of glycogen, a highly branched water-soluble and amorphous storage polyglucan. GlgB was expressed in the be2 be3 double mutant of Arabidopsis that is devoid of BE activity and consequently free of starch. The synthesis of a water-insoluble, partly crystalline, amylose-containing starch-like polyglucan was restored in GlgB-expressing plants, suggesting that BEs origin only have a limited impact on establishing essential characteristics of starch. Moreover, the balance between branching and debranching is crucial for the synthesis of starch, as an excess of branching activity results in the formation of highly branched, water-soluble, poorly crystalline polyglucan.

Single Cells of Pseudomonas aeruginosa Exhibit Electrotaxis and Durotaxis Behaviours

Lee Preiss — 2015-06-05

Pseudomonas aeruginosa is frequently associated with nosocomial infections, including polymicrobial wound infections and the complex biofilm communities that reside within the cystic fibrosis lung. P. aeruginosa utilizes flagellum-mediated motility to approach, attach to, and spread across a surface using a combination of swimming, swarming and twitching (type IV pili extension and retraction) motility. We report an innovative approach to study the motility of single P. aeruginosa cells in microfluidic channels possessing different structural geometry, all with the flexibility of being able to manipulate stiffness gradients and electric fields to investigate changes in motility in response to specific stimuli. P. aeruginosa cells exhibit restricted motility in reduced microchannel spaces, with preferential migration toward a stiffer region in a rigidness gradient of substrate medium and preferential migration toward a positive electrode in presence of a pulsed or successive electric field. This single-cue environmental study using microfluidic technology lays the groundwork for multi-cue experimentation to more faithfully mimic conditions in vivo, demonstrating just some of the advantages of this technique. This study is designed to examine the interplay between surface rigidity, mechanical, and electrical cues to pave the way for improvements in the design of anti-fouling surfaces for biomedical applications and to identify new ways to inhibit bacterial biofilm growth through motility restriction.

Graphene oxide chemically decorated with hybrid Ag-Ru/chitosan nanoparticles: fabrication and properties

Murugan Veerapandian — 2015-07-12

Hybridization of distinct materials into a single nanoplatform is relevant to advance materials properties for functional application such as biosensor platform. We report the synthesis and characterization of nanosheets of graphene oxide decorated with hybrid nanoparticles of silver-ruthenium bipyridine complex (Ag@[Ru(bpy)3]2+) core and chitosan shell. Hybrid nanoparticles were first obtained through a sequential wet-chemical approach using in situ reduction, electrostatic and coordination reaction. Oxygenated functional groups of graphene oxide and abundant amine groups of chitosan layer on the surface of hybrid nanoparticles allowed the functionalization reaction. Changes in intrinsic optical, chemical and structural properties of graphene oxide due to hybrid nanoparticles were studied in depth using spectroscopic techniques and an electron microscope. Electrodes modified with nanosheets of graphene oxide-hybrid nanoparticles retain the biocompatibility and displayed an amplified redox property suitable for a broad range of sensing studies.

Investigation of the Antimicrobial Activity of Soy Peptides by Developing a High Throughput Drug Screening Assay

Rekha Dhayakaran — 2016-03-23

BackgroundAntimicrobial resistance is a great concern in the medical community, as well as food industry. Soy peptides were tested against bacterial biofilms for their antimicrobial activity. A high throughput drug screening assay was developed using microfluidic technology, RAMAN spectroscopy, and optical microscopy for rapid screening of antimicrobials and rapid identification of pathogens.nnMethodsSynthesized PGTAVFK and IKAFKEATKVDKVVVLWTA soy peptides were tested against Pseudomonas aeruginosa and Listeria monocytogenes using a microdilution assay. Microfluidic technology in combination with Surface Enhanced RAMAN Spectroscopy (SERS) and optical microscopy was used for rapid screening of soy peptides, pathogen identification, and to visualize the impact of selected peptides.nnResultsThe PGTAVFK peptide did not significantly affect P. aeruginosa, although it had an inhibitory effect on L. monocytogenes above a concentration of 625 {micro}M. IKAFKEATKVDKVVVLWTA was effective against both P. aeruginosa and L. monocytogenes above a concentration of 37.2 {micro}M. High throughput drug screening assays were able to reduce the screening and bacterial detection time to 4 h. SERS spectra was used to distinguish the two bacterial species.nnConclusionsPGTAVFK and IKAFKEATKVDKVVVLWTA soy peptides showed antimicrobial activity against P. aeruginosa and L. monocytogenes. Development of high throughput assays could streamline the drug screening and bacterial detection process. General significance: The results of this study show that the antimicrobial properties, biocompatibility, and biodegradability of soy peptides could possibly make them an alternative to the ineffective antimicrobials and antibiotics currently used in the food and medical fields. High throughput drug screening assays could help hasten pre-clinical trials in the medical field.

Dual immunosensor based on methylene blue-electroadsorbed graphene oxide for rapid detection of influenza virus antigen

Murugan Veerapandian — 2016-03-23

Rapid detection of influenza viral infections in the poultry farm is advantageous in several facts such as environmental/personal safety, food-security and socio-economy. Herein, we report the development of an electrochemical based dual sensor platform composed of methylene blue-electroadsorbed graphene oxide nanostructures modified with monoclonal antibodies, H5N1 and H1N1. Bio-functional layers comprised of chitosan and protein-A molecules were implemented at the interface of sensor element and antibodies, which synergistically enriched the bio-activity of immobilized antibodies for the immune complex formation. The differential pulse voltammetric signals resulted from the developed immunosensor platform exhibited a good correlation (R2= 0.9978 for H1N1 and R2=0.9997 for H5N1) for the wide range of target concentrations (25 to 500 pM). Chronoamperometric study also revealed the amplified current sensitivity of the immunoelectrodes even at the picomolar level. The proposed immunosensor design not only provide rapid analytical response time (<1 min) but also provide simplicity in fabrication and instrumentation which paves an attractive platform for on-farm monitoring of viral infections.

Compensatory responses by managers, commercial and recreational harvesters to variation in stock abundance of Lake Erie walleye (Sander vitreus vitreus)

Katrine Turgeon — 2016-06-29

Delayed quota adjustments, and/or lagged fishing effort and catch by harvesters, to changes in stock abundance may induce unstable population dynamics and exacerbate the risk of fishery collapse. We examined a 39-y time series of change to quotas by managers, and to effort and catch by both commercial harvesters and anglers, in response to changes in Lake Erie walleye abundance (Sander vitreus) estimated both contemporaneously and retrospectively. Quotas, commercial effort and catch were entrained by contemporaneous estimates of stock abundance. Recreational effort and harvest were not; they had better tracked abundance, as better estimated today, than did the commercial fishery. During the 1990s, a significant mismatch developed between the quota-driven commercial harvest and stock abundance that persisted until a new assessment process obtained. The quasi-open access recreational fishery, instead, freed anglers to respond better to stock abundance. Further elaboration of adaptive risk governance processes, including multi-model inference for stock assessments, may bode well to further reduce risk to fisheries imposed by lagged adjustments to variation in stock abundance.

Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filament

Maureen M Mundia — 2016-08-18

In this study, we utilized mouse hybridoma cell lines stably expressing ectopic wild-type Rad51, or the Rad51-K133A and Rad51-K133R catalytic mutants deficient in ATP binding and ATP hydrolysis, respectively, to investigate effects on the Rad51 nucleoprotein filament in vivo. Immunoprecipitation studies reveal interactions between ectopic wild-type Rad51, Rad51-K133A and Rad51-K133R and endogenous Rad51, Brca2 and p53 proteins. Importantly, the expression of Rad51-K133A and Rad51-K133R catalytic mutants (but not wild-type Rad51) targets endogenous Rad51, Brca2 and p53 proteins for proteasome-mediated degradation. Expression of Rad51-K133R significantly reduces nascent DNA synthesis (3 polymerization) during homologous recombination (HR), but the effects of Rad51-K133A on 3 polymerization are considerably more severe. Provision of additional wild-type Rad51 in cell lines expressing Rad51-K133A or Rad51-K133R does not restore diminished levels of endogenous Brca2, Rad51 or p53, nor restore the deficiency in 3 polymerization. Cells expressing Rad51-K133A are also significantly reduced in their capacity to drive strand exchange through regions of heterology. Our results reveal an interesting mechanistic dichotomy in the way mutant Rad51-K133A and Rad51-K133R proteins influence 3 polymerization and provide novel insight into the mechanism of their dominant-negative phenotypes.

Root hair-endophyte stacking (RHESt) in an ancient Afro-Indian crop creates an unusual physico-chemical barrier to trap pathogen(s)

Walaa K Mousa — 2016-08-25

The ancient African crop, finger millet, has broad resistance to pathogens including the toxigenic fungus Fusarium graminearum. Here we report the discovery of a novel plant defence mechanism, resulting from an unusual symbiosis between finger millet and a root-inhabiting bacterial endophyte, M6 (Enterobacter sp.). Seed-coated M6 swarms towards Fusarium attempting to penetrate root epidermis, induces growth of root hairs which then bend parallel to the root axis, then forms biofilm-mediated microcolonies, resulting in a remarkable, multi-layer root hair-endophyte stack (RHESt). RHESt results in a physical barrier that prevents entry and/or traps F. graminearum which is then killed. Thus M6 creates its own specialized killing microhabitat. M6 killing requires c-di-GMP-dependent signalling, diverse fungicides and xenobiotic resistance. Further molecular evidence suggests long-term host-endophyte-pathogen co-evolution. The end-result of this remarkable symbiosis is reduced DON mycotoxin, potentially benefiting millions of subsistence farmers and livestock. RHESt demonstrates the value of exploring ancient, orphan crop microbiomes.

The dichotomous effects of caffeine on homologous recombination in mammalian cells

Alissa C Magwood — 2016-08-29

This study was initiated to examine the effects of caffeine on the DNA damage response (DDR) and homologous recombination (HR). An initial 2 h exposure to 5 mM caffeine slowed a fraction of the cells in G1, but thereafter, continued caffeine exposure permitted this cell fraction to progress through the cycle until they eventually stalled at G2/M and underwent apoptosis. This prolonged caffeine exposure also induced a strong DDR along with subsequent activation of wild-type p53 protein. An unexpected observation was the caffeine-induced depletion of Rad51 (and Brca2) proteins. Consequently, caffeine-treated cells were expected to be inefficient in HR. However, a dichotomy in the HR response of cells to caffeine treatment was revealed. Caffeine treatment rendered cells significantly better at performing the nascent DNA synthesis that accompanies the early strand invasion steps of HR. Conversely, the increase in nascent DNA synthesis did not translate into a higher level of gene targeting events. Levels of Rad51 appear to be irrelevant. Thus, prolonged caffeine exposure stalls the cell cycle, induces a p53-mediated apoptotic response and a down-regulation of critical HR proteins, and stimulates early steps of HR, but not the formation of complete repair products.

Electroceutical disinfection strategies impair the motility of pathogenic Pseudomonas aeruginosa and Escherichia coli

Doxsee, K. — 2016-11-16

Electrotaxis or galvanotaxis refers to the migration pattern of cells induced in response to electrical potential. Although it has been extensively studied in mammalian cells, electrotaxis has not been explored in detail in bacterial cells; information regarding the impact of current on pathogenic bacteria is severely lacking. Therefore, we designed a series of single and multi-cue experiments to assess the impact of varying currents on bacterial motility dynamics in pathogenic multi-drug resistant (MDR) strains of Pseudomonas aeruginosa and Escherichia coli using a microfluidic platform. Motility plays key roles in bacterial migration and the colonization of surfaces during the formation of biofilms, which are inherently recalcitrant to removal and resistant to traditional disinfection strategies (e.g. antibiotics). Use of the microfluidic platform allows for exposure to current, which can be supplied at a range that is biocidal to bacteria, yet physiologically safe in humans (single cue). This system also allows for multi-cue experiments where acetic acid, a relatively safe compound with anti-fouling/antimicrobial properties, can be combined with current to enhance disinfection. These strategies may offer substantial therapeutic benefits, specifically for the treatment of biofilm infections, such as those found in the wound environment. Furthermore, microfluidic systems have been successfully used to model the unique microfluidic dynamics present in the wound environment, suggesting that these investigations could be extended to more complex biological systems. Our results showed that the application of current in combination with acetic acid has profound inhibitory effects on MDR strains of P. aeruginosa and E. coli, even with brief applications. Specifically, E. coli motility dynamics and cell survival were significantly impaired starting at a concentration of 125 A DC and 0.31% acetic acid, while P. aeruginosa was impaired at 70 A and 0.31% acetic acid. As these strains are relevant wound pathogens, it is likely that this strategy would be effective against similar strains in vivo and could represent a new approach to hasten wound healing.

Multilevel And Sex-Specific Selection On Competitive Traits In North American Red Squirrels

Fisher, D. N. — 2017-05-04

Individuals often interact more closely with some members of the population (e.g. offspring, siblings or group members) than they do with other individuals. This structuring of interactions can lead to multilevel natural selection, where traits expressed at the group-level influence fitness alongside individual-level traits. Such multilevel selection can alter evolutionary trajectories, yet is rarely quantified in the wild, especially for species that do not interact in clearly demarcated groups. We quantified multilevel natural selection on two traits, postnatal growth rate and birth date, in a population of North American red squirrels (Tamiasciurus hudsonicus). The strongest level of selection was typically within-acoustic social neighbourhoods (within 130m of the nest), where growing faster and being born earlier than nearby litters was key, while selection on growth rate was also apparent both within-litters and within-study areas. Higher population densities increased the strength of selection for earlier breeding, but did not influence selection on growth rates. Females experienced especially strong selection on growth rate at the within-litter level, possibly linked to the biased bequeathal of the maternal territory to daughters. Our results demonstrate the importance of considering multilevel and sex-specific selection in wild species, including those that are territorial and sexually monomorphic.nnData archival: the data set is archived on Dryad (info XXX), with a five-year embargo from the date of publication.

The genome sizes of ostracod crustaceans correlate with body size and phylogeny

Jeffery, N. W. — 2017-03-07

Within animals a positive correlation between genome size and body size has been detected in several taxa but not in others, such that it remains unknown how pervasive this pattern may be. Here we provide another example of a positive relationship, in a group of crustaceans whose genome sizes have not previously been investigated. We analyze genome size estimates for 46 species across Class Ostracoda, including 29 new estimates made using Feulgen image analysis densitometry and flow cytometry. Genome sizes in this group range ~80-fold, a level of variability that is otherwise not seen in crustaceans with the exception of some malacostracan orders. We find a strong positive correlation between genome size and body size across all species, including after phylogenetic correction. We additionally detect evidence of XX/XO sex determination in all three species of myodocopids where male and female genome sizes were estimated. On average, genome sizes are larger but less variable in myodocopids than in podocopids, and marine ostracods have larger genomes than freshwater species, but this appears to be explained by phylogenetic inertia. The relationship between phylogeny, genome size, body size, and habitat is complex in this system, and will benefit from additional data collection across various habitats and ostracod taxa.

DNA Metabarcoding For High-Throughput Monitoring Of Estuarine Macrobenthic Communities

Lobo, J. — 2017-06-01

Benthic communities are key components of aquatic ecosystems biomonitoring. However, morphology-based species identifications remain a low-throughput, and sometimes ambiguous, approach. Despite metabarcoding methodologies have been applied for above-species taxa inventories in marine meiofaunal communities, a comprehensive approach providing species-level identifications for estuarine macrobenthic communities is still lacking. Here we report a combination of experimental and field studies demonstrating the aptitude of COI metabarcoding to provide robust species-level identifications within a framework of high-throughput monitoring of estuarine macrobenthic communities. To investigate the ability to recover DNA barcodes from all species present in a bulk DNA extract, we assembled 3 phylogenetically diverse communities, using 4 different primer pairs to generate PCR products of the COI barcode region. Between 78 and 83% of the species in the tested communities were recovered through HTS. Subsequently, we compared morphology and metabarcoding-based approaches to determine the species composition from four distinct sites of an estuary. Our results indicate that the species richness would be considerably underestimated if only morphological methods were used. Although further refinement is required for improving the efficiency and output of this approach, here we show the great aptitude of COI metabarcoding to provide high quality and auditable species identifications in macrobenthos monitoring.

Cellular Interplay and Cytokine Hierarchy Cause Pathological Cardiac Hypertrophy in RAF1-Mutant Noonan Syndrome

Yin, J. C. — 2017-03-30

Noonan syndrome (NS) is caused by mutations in RAS/ERK pathway genes, and is characterized by craniofacial, growth, cognitive and cardiac defects. NS patients with kinase-activating RAF1 alleles typically develop pathological left ventricular hypertrophy (LVH), which is reproduced in Raf1L613V/+ knock-in mice. Here, using inducible Raf1L613V expression, we show that LVH results from the interplay of cardiac cell types. Cardiomyocyte Raf1L613V enhances Ca2+ sensitivity and cardiac contractility without causing hypertrophy. Raf1L613V expression in cardiomyocytes or activated fibroblasts exacerbates pressure overload-evoked fibrosis. Endothelial/endocardial (EC) Raf1L613V causes cardiac hypertrophy without affecting contractility. Co-culture and neutralizing antibody experiments reveal a cytokine (TNF/IL6) hierarchy in Raf1L613V-expressing ECs that drives cardiomyocyte hypertrophy in vitro. Furthermore, post-natal TNF inhibition normalizes the increased wall thickness and cardiomyocyte hypertrophy in vivo. We conclude that NS cardiomyopathy involves cardiomyocytes, ECs, and fibroblasts, TNF/IL6 signaling components represent potential therapeutic targets, and abnormal EC signaling might contribute to other forms of LVH.

Recent Advancement in Biosensors Technology for Animal and Livestock Health Management

Neethirajan, S. — 2017-04-19

The term biosensors encompasses devices that have the potential to quantify physiological, immunological and behavioural responses of livestock and multiple animal species. Novel biosensing methodologies offer highly specialised monitoring devices for the specific measurement of individual and multiple parameters covering an animals physiology as well as monitoring of an animals environment. These devices are not only highly specific and sensitive for the parameters being analysed, but they are also reliable and easy to use, and can accelerate the monitoring process. Novel biosensors in livestock management provide significant benefits and applications in disease detection and isolation, health monitoring and detection of reproductive cycles, as well as monitoring physiological wellbeing of the animal via analysis of the animals environment. With the development of integrated systems and the Internet of Things, the continuously monitoring devices are expected to become affordable. The data generated from integrated livestock monitoring is anticipated to assist farmers and the agricultural industry to improve animal productivity in the future. The data is expected to reduce the impact of the livestock industry on the environment, while at the same time driving the new wave towards the improvements of viable farming techniques. This review focusses on the emerging technological advancements in monitoring of livestock health for detailed, precise information on productivity, as well as physiology and well-being. Biosensors will contribute to the 4th revolution in agriculture by incorporating innovative technologies into cost-effective diagnostic methods that can mitigate the potentially catastrophic effects of infectious outbreaks in farmed animals

Amplified visual immunosensor integrated with nanozyme for ultrasensitive detection of avian influenza virus

Ahmed, S. R. — 2017-04-19

Nanomaterial-based artificial enzymes or nanozymes exhibits superior properties such as stability, cost effectiveness and ease of preparation in comparison to conventional enzymes. However, the lower catalytic activity of nanozymes limits their sensitivity and thereby practical applications in the bioanalytical field. To overcome this drawback, herein we propose a very simple but highly sensitive, specific and low-cost dual enhanced colorimetric immunoassay for the detection of avian influenza virus A (H5N1) through facile in situ synthesis of gold nanoparticles and their peroxidase-like enzymatic activity. 3,3,5,5-Tetramethylbenzidine (TMBZ) was used as a reducing agent to produce gold nanoparticles (Au NPs) from a viral target-specific antibody-gold ion complex. The developed blue color from the sensing design was further amplified through catalytic activity of Au NPs in presence of TMBZ-hydrogen peroxide (H2O2) complex. The developed dual enhanced colorimetric immunosensor enables the detection of avian influenza virus A (H5N1) with a limit of detection (LOD) of 1.11 pg/mL. Our results further confirms that the developed assay has superior sensitivity than the conventional ELISA method, plasmonic-based bioassay and commercial flu diagnostic kits.

FplA From Fusobacterium nucleatum Is A Type Vd Autotransporter Phospholipase With A Proposed Role In Altered Host Signaling And Evasion Of Autophagy

Casasanta, M. A. — 2017-05-10

Fusobacterium nucleatum is a pathogenic oral bacterium that is linked to multiple human infections and colorectal cancer. While most Gram-negative pathogens utilize secretion systems for cellular invasion and infection, F. nucleatum lacks Type I, II, III, IV, and VI secretion. By contrast, F. nucleatum strains are enriched in Type V secreted autotransporters, which are Gram-negative bacterial virulence factors critical for binding and entry into host cells. Here we present the first biochemical characterization of a F. nucleatum Type Vd phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby rename Fusobacterium phospholipase autotransporter (FplA). FplA is expressed as a full-length 85 kDa outer membrane embedded protein, or as a truncated phospholipase domain that remains associated with the outer membrane. Using multiple FplA constructs we characterized lipid substrate specificity, potent inhibitors, and chemical probes to detect and track this enzyme family. While the role of FplA is undetermined in F. nucleatum virulence, homologous phospholipases from intracellular pathogens are critical for vacuole escape, altered host signaling, and intracellular survival. We hypothesize that upon intracellular invasion of the host, FplA could play a role in phagosomal escape, subversion of autophagy, or eicosanoid-mediated inflammatory signaling, as we show that FplA binds with high affinity to host phosphoinositide signaling lipids critical to these processes. Our identification of substrates, inhibitors, and chemical probes for FplA, in combination with an fplA gene deletion strain, encompass a powerful set of tools for the future analysis of FplA in vivo. In addition, these studies will guide the biochemical characterization of additional Type Vd autotransporter phospholipases.nnIMPORTANCEF. nucleatum is an emerging pathogen that is linked to the pathogenesis of colorectal cancer, yet there is a critical knowledge gap in the mechanisms used by this bacterium to elicit changes in the host for intracellular entry and survival. As phospholipases are critical virulence factors for intracellular bacteria to initiate vacuole lysis, cell-to-cell spread, and evasion of autophagy, we set out to characterize a unique Type Vd secreted phospholipase A1 enzyme from F. nucleatum. Our results show a potential role for modulating host signaling pathways through cleavage of phosphoinositide dependent signaling lipids. These studies open the door for further characterization of this unique enzyme family in bacterial virulence, host-pathogen interactions, and for F. nucleatum, in colorectal carcinogenesis.

Nanoscale Imaging Approaches To Quantifying The Electrical Properties Of Pathogenic Bacteria

Berthelot, R. — 2017-05-25

Biofilms are natural, resilient films formed when microorganisms adhere to a surface and form a complex three-dimensional structure that allows them to persist in a wide variety of environments. Readily forming in hospitals and on medical equipment, biofilms are frequent causes of infections and their subsequent complications. Due to the complexity of these structures, systematically studying individual bacterial cells and their interactions with their surrounding environment will provide a deeper understanding of the processes occurring within the biofilm as whole versus bulk population based methods that do not differentiate individual cells or species. Methods based on atomic force microscopy (AFM) are particularly suited to the study of individual cells, but are underutilized for the study of bacterial electrical properties. The ability of electrical currents to impair bacterial attachment is well documented, but to utilize electrical current as an effective antibacterial treatment, it is important to understand the electrical properties of bacteria. Therefore, we used AFM, Kelvin probe force microscopy, and ResiScope to measure the surface potential and conductance of Psuedomonas aeruginosa and methicillin resistance Staphylococcus aureus (MRSA) on gold and stainless steel. This is the first study to directly measure the electrical resistance of single bacterial cells using ResiScope. Our goal was to develop a framework for measuring biological molecules using conductive atomic force microscopy. We found that the average resistance for P. aeruginosa was 135.4{+/-}25.04 G{Omega}, while MRSA had an average of 173.4{+/-}16.28 G{Omega}. Using KPFM, the surface potential of MRSA shifted from -0.304 V to 0.153 V and from -0.280 V to 0.172 V for P. aeruginosa on gold versus stainless steel substrates, respectively. In an attempt to identify a potential charge carrier, peptidoglycan was also measured with the ResiScope module and shown to have a resistance of 105 G{Omega}.

Rapid Detection of Norovirus Using Paper-based Microfluidic Device

Weng, X. — 2017-07-12

Noroviruses (NoV) are the leading cause of outbreak of acute gastroenteritis worldwide. A substantial effort has been made in the development of analytical devices for rapid and sensitive food safety monitoring via the detection of foodborne bacteria, viruses and parasites. Conventional analytical approaches for noroviruses suffer from some critical weaknesses: labor-intensive, time-consuming, and relatively low sensitivity. In this study, we developed a rapid and highly sensitive biosensor towards point-of-care device for noroviruses based on 6-carboxyfluorescein (6-FAM) labeled aptamer and nanomaterials, multi-walled carbon nanotubes (MWCNTs) and graphene oxide (GO). In an assay, the fluorescence of 6-FAM labeled aptamer was quenched by MWCNTs or GO via fluorescence resonance energy transfer (FRET). In the presence of norovirus, the fluorescence would be recovered due to the release of the 6-FAM labeled aptamer from MWCNTs or GO. An easy-to-make paper-based microfluidic platform made by nitrocellulose membrane was used to conduct the assay. The quantitative detection of norovirus virus-like particles (NoV VLPs) was successfully performed. A linear range of 0-12.9 g/mL with a detection limit of 40 pM and 30 pM was achieved for the MWCNTs and GO based paper sensors, respectively. The results suggested the developed paper-based microfluidic device is simple, cost-effective and holds the potential of rapid in situ visual determination for noroviruses with remarkable sensitivity and specificity, which provides a new way for early identification of NoV and thereby an early intervention for preventing the spread of an outbreak.

Self-assembled Star-shaped Chiroplasmonic Gold Nanoparticles for Ultrasensitive Chiro-immunosensor of Viruses

Ahmed, S. R. — 2017-07-12

Near field optics and optical tunneling light-matter interaction in the superstructure of chiral nanostructures and semiconductor quantum dots exhibit strong optical rotation activity that may open a new window for chiral-based bioanalytes detection. Herein we report an ultrasensitive, chiro-immunosensor using superstructure of chiral gold nanohybrids (CAu NPs) and quantum dots (QDs). Self-assembling techniques were employed to create asymmetric plasmonic chiral nanostructures for extending the spectral range of circular dichroism (CD) response for obtaining superior plasmonic resonant coupling with the QDs excitonic state; this may help to achieve lower the limit of detection (LOD) values. As a result, the designed probe exhibited avian influenza A (H5N1) viral concentration at picomolar level, a significant improvement in sensitivity in comparison to a non-assembled CAu NPs based chiroassay. The practicability of the proposed sensing system was successfully demonstrated on several virus cultures including, avian influenza A (H4N6) virus, fowl adenovirus and coronavirus in blood samples. The results of our study highlights that exciton-plasmon interaction changes chirality and the self-assembled nanostructures are an efficient strategy for enhancing the sensitivity of plasmonic nanosensors.

Paper based microfluidic aptasensor for food safety

Weng, X. — 2017-07-12

Food analysis is requiring rapid, accurate, sensitive and cost-effective methods to monitor and guarantee the safety and quality to fulfill the strict food legislation and consumer demands. In our study, a nano-materials enhanced multipurpose paper based microfluidic aptasensor was demonstrated as a sensing tool for accurate detection of food allergens and food toxins. Graphene oxide (GO) and specific aptamer-functionalized quantum dots (QDs) were employed as probes, the fluorescence quenching and recovering of the QDs caused by the interaction among GO, aptamer-functionalized QDs and the target protein were investigated to quantitatively analyze the target concentration. The homogenous assay was performed on the paper based microfluidic chip, which significantly decreased the sample and reagent consumptions and reduced the assay time. Egg white lysozyme, {beta}-conglutin lupine and food toxins, okadaic acid and brevetoxin standard solutions and spiked food samples were successfully assayed by the presented aptasensor. Dual-target assay was completed within 5 min, and superior sensitivities were achieved when testing the samples with commercial ELISA kits side by side.

Chitozyme: First Peroxidase-like Activity of Chitosan for Multiplexed Visual Detection of H2O2, Glucose and Lactate on Paper-based Device

Ahmed, S. R. — 2017-07-12

Visual read-out diagnostics tools are promising candidates for field applicable medical devices. Current colorimetric biosensors require introduction of natural enzymes or nanozymes, which has some serious drawbacks for practical applications. Chitosan, a natural polymer, provides safe and efficient compound in medical and pharmaceutical technology. Herein, we report on a simple, cost-efficient, field-portable, environmental friendly and ultra-sensitive multiplex detection platform based on peroxidase-like activity of chitosan in the presence of 3,3,5,5-Tetramethylbenzidine (TMBZ) and H2O2. This straight forward signal amplification strategy was successfully applied to detect H2O2, glucose and lactate with the limit of detection (LOD) of 2.64 pM, 0.104 M and 2.8 nM respectively, represents the lowest LOD of H2O2, glucose and lactate with visual read-out. The chitosan-based assay performance was also retained in complex biological media for glucose and lactate detection. Furthermore, the proposed assay was successfully demonstrated as a paper-based colorimetric biosensor. Most importantly, the simplicity, biocompatibility and sensitivity of the proposed assay will open new doors for instrument free naked eye visual detection of H2O2, glucose and lactate detection.

Climbing Legumes: An Underutilized Resource With Significant Potential to Intensify Farming on Terrace Walls (FTW) for Smallholder Farmers

Clark, J. C. — 2017-09-05

Millions of subsistence farmers cultivate crops on terraces. These farmers face unique challenges including severe shortages of arable land and remoteness leading to poor access to inputs including nitrogen fertilizer. These challenges contribute to human and livestock malnutrition. Terrace walls (risers) as a vertical surface to grow climbing or trailing legumes represents an opportunity to help overcome these challenges. These crops are rich in minerals and protein, and their associated microbes produce nitrogen fertilizer. Rice bean is already grown on terrace risers in South Asia. This paper reviews the literature concerning crops that are currently farmed on terrace walls (FTW), then surveys climbing legume species that have potential for FTW, focusing on crops that are nutritious and tolerate shade (caused by the terrace wall) and resist drought (many terrace farms experience an extended dry season). A total of 29 legume species are discussed including climbing varieties of jack bean, common bean, cowpea, winged bean, horse gram and velvet bean. The review concludes by discussing the practical challenges of farmer adoption of FTW and makes concrete recommendations. Terrace wall cultivation of legumes represents an opportunity to intensify agriculture and increase resiliency in remote mountainous areas.

A Sequel to Sanger: Amplicon Sequencing That Scales

Hebert, P. D. — 2017-09-20

Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system. By examining templates from more than 5,000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL reduces costs 40-fold from Sanger analysis. Reflecting the capacity of each instrument to recover sequences from more than five million DNA extracts a year, this platform facilitates massive amplicon characterization.

Expedited Assessment of Terrestrial Arthropod Diversity by Coupling Malaise Traps with DNA Barcoding

deWaard, J. R. — 2017-09-25

O_LIMonitoring changes in terrestrial arthropod communities over space and time requires a dramatic increase in the speed and accuracy of processing samples that cannot be achieved with morphological approaches.nC_LIO_LIThe combination of DNA barcoding and Malaise traps allows expedited, comprehensive inventories of species abundance whose cost will rapidly decline as high-throughput sequencing technologies advance.nC_LIO_LIAside from detailing protocols from specimen sorting to data release, this paper describes their use in a survey of arthropod diversity in a national park that examined 20,000 specimens representing 2200 species.nC_LIO_LIThese protocols can support arthropod monitoring programs at regional, national, and continental scales.nC_LI

Traditional Norwegian Kveik Yeasts: Underexplored Domesticated Saccharomyces cerevisiae Yeasts

Preiss, R. — 2017-09-27

Human activity has resulted in the domestication of Saccharomyces cerevisiae yeasts specifically adapted to beer production. While there is evidence beer yeast domestication was accelerated by industrialization of beer, there also exists a home-brewing culture in western Norway which has passed down yeasts referred to as kveik for generations. This practice has resulted in ale yeasts which are typically highly flocculant, phenolic off flavour negative (POF-), and exhibit a high rate of fermentation, similar to previously characterized lineages of domesticated yeast. Additionally, kveik yeasts are highly temperature tolerant, likely due to the traditional practice of pitching yeast into warm (>30 {degrees}C) wort. Here, we characterize kveik yeasts from 9 different Norwegian sources via PCR fingerprinting, phenotypic screens, lab-scale fermentations, and flavour metabolite analysis using HS-SPME-GC-MS. Genetic fingerprinting via interdelta PCR suggests that kveik yeasts form a lineage distinct from other domesticated yeasts. Our analyses confirm that kveik yeasts display hallmarks of domestication such as loss of 4-vinylguaiacol production and high flocculation, and show superior thermotolerance, ethanol tolerance, fermentation rate, and unique flavour metabolite production profiles in comparison to other ale strains, suggesting a broad industrial potential for this group of yeasts.

Spatial correlation as an early warning signal of regime shifts in a multiplex disease-behaviour network

Jentsch, P. C. — 2017-09-29

Early warning signals of sudden regime shifts are a widely studied phenomenon for their ability to quantify a systems proximity to a tipping point to a new and contrasting dynamical regime. However, this effect has been little studied in the context of the complex interactions between disease dynamics and vaccinating behaviour. Our objective was to determine whether critical slowing down (CSD) occurs in a multiplex network that captures opinion propagation on one network layer and disease spread on a second network layer. We parameterized a network simulation model to represent a hypothetical self-limiting, acute, vaccine-preventable infection with shortlived natural immunity. We tested five different network types: random, lattice, small-world, scale-free, and an empirically derived network. For the first four network types, the model exhibits a regime shift as perceived vaccine risk moves beyond a tipping point from full vaccine acceptance and disease elimination to full vaccine refusal and disease endemicity. This regime shift is preceded by an increase in the spatial correlation in non-vaccinator opinions beginning well before the bifurcation point, indicating CSD. The early warning signals occur across a wide range of parameter values. However, the more gradual transition exhibited in the empirically-derived network underscores the need for further research before it can be determined whether trends in spatial correlation in real-world social networks represent critical slowing down. The potential upside of having this monitoring ability suggests that this is a worthwhile area for further research.

Global Land Use Implications of Dietary Trends: A Tragedy of the Commons

Rizvi, S. — 2017-09-28

Global food security and agricultural land management represent two urgent and intimately related challenges that humans must face in the coming decades. Here, we quantify the changes in the global agricultural land footprint if the world were to adhere to the dietary guidelines put forth by the United States Department of Agriculture (USDA), while accounting for the land use change incurred by import/export required to meet those guidelines. We analyze data at country, continent, and global levels. USDA guidelines are viewed as an improvement on the current land-intensive diet of the average American, but despite this our results show that global adherence to USDA guidelines would require up to 1 gigahectare of additional agricultural land--roughly the size of Canada. The results also show a strong divide between Eastern and Western hemispheres. Because countries increasingly import most of their food, meeting USDA guidelines could cause a Tragedy of the Commons, where self-interested actors race to over-exploit the shared resource of global agricultural lands. National dietary guidelines and practices thus need to be coordinated internationally in order to spare our remaining natural lands, in much the same way that countries are coordinating greenhouse gas emissions.

Interconnections accelerate collapse in a socio-ecological metapopulation

Dockstader, Z. — 2017-09-28

Resource over-exploitation can have profound effects on both ecosystems and the human populations residing in them. Models of population growth based on a depletable resources have been studied previously, but relatively few consider metapopulation effects. Here we analyze a socio-ecological metapopulation model where resources grow logistically on each patch. Each population harvests resources on its own patch to support population growth, but can also harvest resources from other patches when their own patch resources become scarce. We find that allowing populations to harvest from other patches significantly accelerates collapse and also increases the parameter regime for which collapse occurs, compared to a model where populations are not able to harvest resources from other patches. As the number of patches in the metapopulation increases, collapse is more sudden, more severe, and occurs sooner. These effects also persist under scenarios of asymmetry and inequality between patches. We conclude that metapopulation effects in socio-ecological systems can be both helpful and harmful and therefore require urgent study.

A helicase-containing module defines a family of pCD630-like plasmids in Clostridium difficile

Smits, W. K. — 2017-10-11

Clostridium difficile is a Gram-positive and sporulating enteropathogen that is a major cause of healthcare-associated infections. Even though a large number of genomes of this species have been sequenced, only a few plasmids have been described in the literature. Here, we use a combination of in silico analyses and laboratory experiments to show that plasmids are common in C. difficile. We focus on a group of plasmids that share similarity with the plasmid pCD630, from the reference strain 630. The family of pCD630-like plasmids is defined by the presence of a conserved putative helicase that is likely part of the plasmid replicon. This replicon is compatible with at least some other C. difficile replicons, as strains can carry pCD630-like plasmids in addition to other plasmids. We find two distinct sub-groups of pCD630-like plasmids that differ in size and accessory modules. This study is the first to describe a family of plasmids in C. difficile.

Attending to what and where: Background connectivity integrates categorical and spatial attention

Tompary, A. — 2017-10-06

Top-down attention prioritizes the processing of goal-relevant information throughout visual cortex, based on where that information is found in space and what it looks like. Whereas attentional goals often have both spatial and featural components, most research on the neural basis of attention has examined these components separately. This may reflect the fact that attention is typically studied in individual visual areas that preferentially code for either spatial locations or particular features. Here we investigated how these attentional components are integrated by examining the attentional modulation of functional connectivity between visual areas with different selectivity. Specifically, we used fMRI to measure temporal correlations between spatially-selective regions of early visual cortex and category-selective regions in ventral temporal cortex while participants performed a task that benefitted from both spatial and categorical attention. We found that categorical attention modulated the connectivity of category-selective areas, but only with retinotopic areas that coded for the spatially attended location. The reverse was not true, however, with spatial attention modulating the connectivity of retinotopic areas with category-selective areas coding for both attended and unattended features. This pattern of results suggests that attentional modulation of connectivity is dominated by spatial selection, which in turn gates featural biases. Combined with exploratory analyses of frontoparietal areas that track these changes in connectivity among visual areas, this study begins to shed light on how different components of attention are integrated in support of more complex behavioral goals.

Comprehensive skin microbiome analysis reveals the uniqueness of human-associated microbial communities among the class Mammalia

Ross, A. A. — 2017-10-11

Skin is the largest organ of the body and represents the primary physical barrier between mammals and their external environment. The objective of this research was to generate a skin microbiota baseline for members of the class Mammalia, testing the effects of host species, geographic location, body region, and biological sex. The back, torso, and inner thigh regions of 177 non-human mammals were collected to include representatives from 38 species and 10 mammalian orders. Animals were collected from local farms, zoos, households, and the wild. All samples were amplified using the V3-V4 16S rRNA gene region and sequenced using a MiSeq (Illumina). For reference, previously published skin microbiome data from 20 human participants, sampled using an identical protocol to the non-human mammals, were included in the analysis. Human skin was significantly less diverse than all other mammalian orders and the factor most strongly associated with community variation for all samples was whether the host was a human. Within non-human samples, host taxonomic order was the most significant factor influencing the skin community, followed by the geographic location of the habitat. By comparing the congruence between known host phylogeny and microbial community dendrograms, we observed that Artiodactyla (even-toed ungulates) and Perissodactyla (odd-toed ungulates) had significant congruence, providing first evidence of phylosymbiosis between skin communities and their hosts.nnSignificanceSkin forms a critical protective barrier between a mammal and its external environment. Baseline data on the mammalian skin microbiome is crucial for making informed decisions related to veterinary research and biodiversity conservation strategies, in addition to providing insight into mammalian evolutionary history. To our knowledge, this study represents the largest mammalian skin microbiota project to date. These findings demonstrate that human skin is distinct, not only from other Primates, but from all 10 mammalian orders sampled. Using phylosymbiosis analysis, we provide the first evidence that co-evolution may be occurring between skin communities and their mammalian hosts, which warrants more in-depth future studies of the relationships between mammals and their skin microbiota.

Rapid assessment of phytoplankton assemblages using Next Generation Sequencing - Barcode of Life database: a widely applicable toolkit to monitor biodiversity and harmful algal blooms (HABs)

Ivanova, N. V. — 2019-12-12

Harmful algal blooms have important implications for the health, functioning and services of aquatic ecosystems. Our ability to detect and monitor these events is often challenged by the lack of rapid and cost-effective methods to identify bloom-forming organisms and their potential for toxin production, Here, we developed and applied a combination of DNA barcoding and Next Generation Sequencing (NGS) for the rapid assessment of phytoplankton community composition with focus on two important indicators of ecosystem health: toxigenic bloom-forming cyanobacteria and impaired planktonic biodiversity. To develop this molecular toolset for identification of cyanobacterial and algal species present in HABs (Harmful Algal Blooms), hereafter called HAB-ID, we optimized NGS protocols, applied a newly developed bioinformatics pipeline and constructed a BOLD (Barcode of Life Data System) 16S reference database from cultures of 203 cyanobacterial and algal strains representing 101 species with particular focus on bloom and toxin producing taxa. Using the new reference database of 16S rDNA sequences and constructed mock communities of mixed strains for protocol validation we developed new NGS primer set which can recover 16S from both cyanobacteria and eukaryotic algal chloroplasts. We also developed DNA extraction protocols for cultured algal strains and environmental samples, which match commercial kit performance and offer a cost-efficient solution for large scale ecological assessments of harmful blooms while giving benefits of reproducibility and increased accessibility. Our bioinformatics pipeline was designed to handle low taxonomic resolution for problematic genera of cyanobacteria such as the Anabaena-Aphanizomenon-Dolichospermum species complex, two clusters of Anabaena (I and II), Planktothrix and Microcystis. This newly developed HAB-ID toolset was further validated by applying it to assess cyanobacterial and algal composition in field samples from waterbodies with recurrent HABs events.

coil: an R package for cytochrome C oxidase I (COI) DNA barcode data cleaning, translation, and error evaluation.

Nugent, C. M. — 2019-12-12

Biological conclusions based on DNA barcoding and metabarcoding analyses can be strongly influenced by the methods utilized for data generation and curation, leading to varying levels of success in the separation of biological variation from experimental error. The five-prime region of cytochrome c oxidase subunit I (COI-5P) is the most common barcode gene for animals, with conserved structure and function that allows for biologically informed error identification. Here, we present coil (https://CRAN.R-project.org/package=coil), an R package for the pre-processing and error assessment of COI-5P animal barcode and metabarcode sequence data. The package contains functions for placement of barcodes into a common reading frame, accurate translation of sequences to amino acids, and highlighting insertion and deletion errors. The analysis of 10,000 barcode sequences of varying quality demonstrated how coil can place barcode sequences in reading frame and distinguish sequences containing indel errors from error-free sequences with greater than 97.5% accuracy. Package limitations were tested through the analysis of COI-5P sequences from the plant and fungal kingdoms as well as the analysis of potential contaminants: nuclear mitochondrial pseudogenes and Wolbachia COI-5P sequences. Results demonstrated that coil is a strong technical error identification method but is not reliable for detecting all biological contaminants.

Effect of extracorporeal shock wave therapy on equine umbilical cord blood mesenchymal stromal cells in vitro

Salcedo-Jimenez, R. — 2020-01-11

Extracorporeal shock wave therapy (ESWT) has been shown to induce different biological effects on a variety of cells, including regulation and stimulation of their function and metabolism. ESWT can promote different biological responses such as proliferation, migration, and regenerations of cells. Recent studies have shown that mesenchymal stromal cells (MSCs) secrete factors that enhance the regeneration of tissues, stimulate proliferation and differentiation of cells and decrease inflammatory and immune-reactions. Clinically, the combination of these two therapies has been used as a treatment for tendon and ligament lesions in horses; however, there is no scientific evidence supporting this combination of therapies in vivo. Therefore, the objectives of the study were to evaluate the effects of ESWT on equine umbilical cord blood mesenchymal stromal cells (CB-MSCs) proliferative, metabolic, migrative, differentiation, and immunomodulatory properties in vitro. Three equine CB-MSC cultures from independent donors were treated using an electrohydraulic shock wave generator attached to a water bath. All experiments were performed as triplicates. Proliferation, viability, migration and immunomodulatory properties of the cells were evaluated. Equine CB-MSCs were induced to evaluate their trilineage differentiation potential. ESWT treated cells had increased metabolic activity, showed positive adipogenic, osteogenic, and chondrogenic differentiation, and showed higher potential for differentiation towards the adipogenic and osteogenic cell fates. ESWT treated cells showed similar immunomodulatory properties to none-ESWT treated cells. Equine CB-MSCs are responsive to ESWT treatment and showed increased metabolic, adipogenic and osteogenic activity, but unaltered immunosuppressive properties. In vivo studies are warranted to determine if synergistic effects occur in the treatment of musculoskeletal injuries if ESWT and equine CB-MSC therapies are combined.

Captive-reared migratory monarch butterflies show natural orientation when released in the wild

Wilcox, A. A. E. — 2020-01-25

Eastern North American migratory monarch butterflies (Danaus plexippus) have faced sharp declines over the last two decades. Although captive rearing has been used as an important tool for engaging the public and supplementing conservation efforts, a recent study that tested monarchs in a flight simulator suggested that captive-reared monarchs lose their capacity to orient southward during fall migration to their Mexican overwintering sites. We raised offspring of wild-caught monarchs on swamp milkweed (Asclepias incarnata) and, after eclosion, individuals were either tested in a flight simulator or radio-tracked in the wild using array of over 100 automated telemetry towers. While only 33% (7/39) of monarchs tested in the flight simulator showed strong southeast to southwest orientation, 97% (28/29) of the radio-tracked individuals were detected by automated towers south or southeast of the release site, up to 200 km away. Our results suggest that, though captive rearing of monarch butterflies may cause temporary disorientation, proper orientation is likely re-established after exposure to natural skylight cues.

Acute oral toxicity and risks of exposure to the neonicotinoid thiamethoxam, and other classes of systemic insecticide, for the Common Eastern Bumblebee (Bombus impatiens)

Mundy-Heisz, K. A. — 2020-01-28

The Common Eastern Bumblebee (Bombus impatiens) is native to North America with an expanding range across Eastern Canada and the USA. This species is commercially produced primarily for greenhouse crop pollination and is a common and abundant component of the wild bumblebee fauna in agricultural, suburban and urban landscapes. However, there is a dearth of pesticide toxicity information about North American bumblebees. The present study determines the acute oral toxicity (48-hour LD50) of cyantraniliprole (>0.54 g/bee), flupyradifurone (>1.7 g/bee), sulfoxaflor (0.0194 g/bee), and thiamethoxam (0.0012 g/bee). Compared with published honey bee (Apis mellifera) LD50 values, the present study shows that thiamethoxam and sulfoxaflor are 4.2x and 7.5x more acutely toxic to B. impatiens, whereas flupyradifurone is more acutely toxic to A. mellifera. The current rule of thumb for toxicity extrapolation beyond the honey bee as a model species, termed 10x safety factor, may be sufficient for bumblebee acute oral toxicity. A comparison of three risk assessment equations suggested that the Standard Risk Approach (SRA) and Fixed Dose Risk Approach (FDRA) provide more nuanced levels of risk evaluation compared to the European Plant Protection Organization (EPPO) Hazard Quotient (HQ), primarily because SRA and FDRA take into account real world variability in pollen and nectar pesticide residues and the chances that bees are exposed to them.

Basal media optimization for the micropropagation and callogenesis of Cannabis sativa L.

Page, S. R. G. — 2020-02-09

Micropropagation of Cannabis sativa is an emerging area for germplasm storage and large-scale production of clean plants. Existing protocols use a limited number of genotypes and are often not reproducible. Previous studies reported MS + 0.5 M TDZ to be optimal for Cannabis nodal micropropagation, yet our preliminary studies using nodal explants suggested this media may not be optimal. It resulted in excessive callus formation, hyperhydricity, low multiplication rates, and high mortality rates. Following an initial screen of four commonly used basal salt mixtures (MS, B5, BABI, and DKW), we determined that DKW produced the healthiest plants. In a second experiment, the multiplication rate and canopy area of explants grown on MS + 0.5 M TDZ and DKW + 0.5 M TDZ were compared using five drug-type cultivars to determine if the preference for DKW was genotype-dependent. Four cultivars had significantly higher multiplication rates on DKW + 0.5 M TDZ with the combined average being 1.5x higher than explants grown on MS + 0.5 M TDZ. The canopy area was also significantly larger on DKW + 0.5 M TDZ for four cultivars with the combined average being twice as large as the explants grown on MS + 0.5 M TDZ. In the third experiment, callogenesis was compared using a range of 2,4-D concentrations (0-30 M) on both MS and DKW and similarly, callus growth was superior on DKW. This study presents the largest comparison of basal salt compositions on the micropropagation of five commercially grown Cannabis cultivars to date.

A novel role for cortical acetylcholine in object memory updating

Jardine, K. H. — 2020-02-08

Reactivated long-term memories can become labile and sensitive to modification. Memories in this destabilized state can be weakened or strengthened, but there is limited research characterizing the mechanisms underlying retrieval-induced qualitative updates (i.e., information integration). We have previously implicated cholinergic transmission in object memory destabilization. Here we present a novel rodent paradigm developed to assess the role of this cholinergic mechanism in qualitative memory updating. The post-reactivation object memory modification (PROMM) task exposes rats to contextual information following object memory reactivation. Subsequent object exploratory performance suggests that the contextual information is integrated with the original memory in a reactivation- and time-dependent manner. This effect is blocked by interference with M1 muscarinic receptors and several downstream signals in perirhinal cortex. These findings therefore demonstrate a hitherto unacknowledged cognitive function for acetylcholine with important implications for understanding the dynamic nature of long-term memory storage in the normal and aging brain.

Height differences in clonal stands of Tussilago farfara promote outcrossing by influencing pollinator behaviour

Dolezal, A. J. — 2020-02-19

Plants with mixed mating systems balance the advantages of selfing and the costs of inbreeding. Previous studies have shown that plant species with the ability to self-pollinate and cross pollinate have strategies to promote outcrossing to increase genetic diversity. Various features of floral morphology are thought to be deliberate mechanisms to manipulate plant mating systems. I hypothesized that within-plant variation in flower stem height is a morphological trait that can reduce geitonogamy by increasing pollinator movement among plants. This hypothesis was tested using coltsfoot (Tussilago farfara); coltsfoot plants host several flowering stems that differ in height, with each stem having a single, terminal flowerhead. I used cut flowers to create populations of coltsfoot in which each plant had four inflorescences with equal or unequal flower stalk lengths and measured frequency of insect pollinators that would stay among flowers within a plant or move to other plants. All pollinators (bee flies, hover flies, solitary bees and wasps) showed a marked discrimination in favor of leaving plants with flowers of different heights and stayed when plants had flowers of the same height. This study shows that variation in flower heights is important for reducing geitonogamy in coltsfoot and suggests that the evolution of this morphological trait should be considered in studies of mating systems.

Propylene glycol-based antifreeze as an effective preservative for DNA metabarcoding of benthic arthropods

Robinson, C. V. — 2020-02-28

Maintaining the integrity of DNA in bulk environmental samples from source to laboratory is crucial for capturing the true range of taxa present within an ecosystem. Preservation consideration of DNA is particularly important if samples are being collected in remote areas and by non-specialist citizen scientists in nationwide programs. Traditionally, absolute ethanol is used as the preferred preservative for environmental samples collected for downstream DNA metabarcoding analyses. However, transport, shipping and DNA extraction of samples preserved in ethanol is a lengthy procedure due to safety restrictions and the requirement of full ethanol evaporation prior to extraction. We examined the efficacy of an easily accessible, non-toxic propylene glycol-based antifreeze as an alternative to absolute ethanol for preserving macroinvertebrate DNA from bulk-benthos DNA samples. We tested the differences in both cytochrome oxidase I (COI) exact sequence variants (ESVs) and COI taxonomic orders detected in both ethanol and antifreeze samples using two processing methods (no evaporation of preservative versus full evaporation). In addition, we assessed the detection of families and genera within the Arthropoda phylum for preservative type, site and processing method. Our results suggest that antifreeze is a suitable alternative to ethanol, a greater global ESV richness reported for antifreeze samples. Additionally, a higher proportion of arthropod reads in ESVs were detected in antifreeze (average 69%) compared with ethanol (average 53%). Finally, antifreeze samples produced similar results for the different processing methods, whereas ethanol samples failed to produce similar results without prior evaporation. Although ethanol is currently widely used for DNA preservation, our results demonstrate that by using antifreeze, it is possible to achieve similar taxonomic coverage and community assemblages of bulk-benthos DNA samples for macroinvertebrates, with the added simplicity and shorter laboratory processing time achieved using an easily available, unregulated preservative.

Cardiac CapZ regulation during acute exercise

Laskary, A. — 2020-03-05

PurposeExercise requires a rapid cardiac response to maintain cardiovascular function. CapZ is a critical stress-response protein in cardiac myocytes. While its role in the pathological stress response has been explored, its part in the physiological response to exercise is unknown. This study examined CapZ regulation during acute exercise and sought to determine its importance in the cardiac response to exercise. MethodsWildtype or cardiac CapZ-deficient transgenic mice were subjected to 20 min of swimming, or exhaustive exercise protocols. Time to exhaustion was a measurement of exercise capacity. Following submaximal exercise, cardiac myofilaments were isolated and probed for CapZ and key regulatory proteins. Myofilament function was assessed using an actomyosin MgATPase assay and total protein phosphorylation quantified with ProQ Diamond staining. Myofilament regulatory proteins following submaximal exercise were quantified by immunoblotting. ResultsTotal myofilament CapZ was unaffected by exercise but increased total CapZIP and decreased phosphorylated CapZIP indicated weakened CapZ-actin interaction. CapZ-deficient transgenic myofilaments lacked changes in CapZIP but BAG3 increased 10%. Time to exhaustion was lower in CapZ-deficient mice in both swimming and running protocols. Actomyosin MgATPase activity was maintained in wildtype mice and impaired with CapZ deficiency. Exercise increased the phosphorylation of several myofilament proteins in wildtype mice but not transgenic animals. Exercise-dependent Increases in myofilament PKC- and -{varepsilon} were mitigated in CapZ-deficient mice. Tcap levels decreased 39 {+/-} 8% in CapZ-deficient myofilaments with exercise and leiomodin 2 increased 78 {+/-} 28% in wildtype myofilaments. ConclusionsCardiac CapZ is a critical player in the physiological response to exercise. CapZ-actin binding is rapidly altered with exercise. Decreased cardiac CapZ limits exercise capacity, impairs myofilament regulation, and leads to a less stable contractile apparatus.

Validating a semi-quantitative method for assessment of degree of methylene blue staining in sentinel lymph nodes

Ram, A. S. — 2020-03-24

Purpose To develop a digital algorithm and validate a semi-quantitative scoring method for surface methylene blue (MB) staining in whole lymph nodes (LN).Methods Lymph nodes from canine models undergoing sentinel lymph node (SLN) mapping were prospectively assessed ex vivo and photographed. Two blinded observers evaluated all images and assigned a semi-quantitative score based on surface staining (0 – no blue stain, 1 – 1-50% stained, 2 – 51-100% stained). A standard reference for degree of blue staining was based on signal-to-background ratios using computer-based imaging software with an output measurement of percentage of staining of the LN. Agreement between observers was assessed using the Kappa coefficient.Results 124 lymph nodes were included and demonstrated strong agreement (K = 0.8007, p < 0.0001) between results of semi-quantitative scoring and image analysis. Also, strong interobserver and intraobserver agreement was observed for the scoring system (K = 0.8051, p < 0.0001 and K = 0.9493, p < 0.0001, respectively).Discussion Agreement between the observer-based scoring system and imaging software illustrates a validated method in assessing MB staining, without the need for analysis software. The use of a semi-quantitative scoring system shows promise for a simple, objective assessment of MB staining in surgery and for future study. Lymph nodes can have variable surface colour, which can make assessment of blue staining challenging for novice observers in certain cases. This study describes a digital algorithm for quantitative analysis of blue staining in LN thereby providing a novel and objective reporting mechanism in scientific research involving SLN mapping.Competing Interest StatementThe authors have declared no competing interest.View Full Text

The effects of Hurricane Otto on the soil ecosystems of three forest types in the Northern Zone of Costa Rica

Eaton, W. — 2020-03-19

Hurricanes rapidly deposit large amounts of canopy material onto tropical forest floors, stimulating metabolic processes involved in the decomposition of these materials and production of N and C resources into the food web. However, little is known about the effects that hurricanes have on specific soil microbial taxa or functional groups involved in these processes. The objectives of this study were to determine how Hurricane Otto influenced three different tropical forest soil ecosystems within the first 8 months after causing damage to a tropical forest by assessing the soil C and N factors and how the soil bacterial and fungal community compositions differed before and after the hurricane. Soil samples were collected from five 2000 m2 permanent plots in Lowland, Upland and Riparian forest systems within the same area in the Northern Zone of Costa Rica. Standard methods were used to measure the amounts Total N, NO3-, NH4+, Total organic C, and Biomass C, while Illumina MiSeq methods were used to generate bacterial and fungal sequences. All data were analyzed using univariate and multivariate statistical methods. Using this "before and after" study design, it was found that the levels of the inorganic N and Biomass C were greater in the Post-Hurricane soil samples. The mean proportion of DNA sequences of complex C degrading/lignin degrading, NH4+-producing, and ammonium oxidizing bacteria, and the complex C degrading/wood rot/lignin degrading and ectomycorrhizal fungi also were greater in the Post-Hurricane soils. We also provide evidence that the excessive amounts of canopy leaf litter and woody debris deposited on the forest floor during Hurricane Otto appears to be selecting for genera that become more dominant Post-Hurricane, perhaps because they may be better able to rapidly process the newly deposited C and N-rich canopy material. This is a rare "before and after" a natural hurricane design that warrants further monitoring.

Conductance mechanisms of rapidly desensitizing cation channelrhodopsins from cryptophyte algae

Sineshchekov, O. A. — 2020-03-21

Channelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. "Bacteriorhodopsin-like" cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, BCCR sequences match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs, which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption and Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function. IMPORTANCECation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-vis spectroscopy and photoelectrophysiology reveal the desensitization is caused by rapid accumulation of a non-conductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

Acute stressors experienced by layer breeders do not affect measures of stress and fear in their offspring

Roedel Lopez Vieira Peixoto, M. — 2020-03-23

Stressors experienced by layer breeders during egg production can lead to changes in the egg hormone content, potentially impacting their offspring, the commercial layers. Genetic differences might also affect the offsprings susceptibility to maternal experiences. In this study, we tested if maternal stress affects measures of stress and fear in five strains of layer breeders: commercial brown 1 & 2, commercial white 1 & 2 and a pure line White Leghorn. Each strain was equally separated into two groups: "Maternal Stress" (MS), where hens were subjected to a series of 8 consecutive days of acute psychological stressors, and "Control," which received routine husbandry. Additional eggs from Control were injected either with corticosterone diluted in a vehicle solution ("CORT") or just "Vehicle." Stress- and fear-responses of the offspring were measured in a plasma corticosterone test and a combined human approach and novel object test. Both MS and CORT treatments failed to affect the measured endpoints in the offspring, but significant strain differences were found. The offspring of the white strains showed a higher physiological response compared to brown strains, but the White 2 offspring was consistently the least fearful strain in the behaviour tests. Our study found that the acute psychological stressors experienced by layer breeders did not affect the parameters tested in their offspring and that corticosterone does not seem to be the primary mediator of maternal stress in laying hens. This is highly important, as in poultry production, layer breeders are often subjected to short-term stressors. In addition, we successfully dissociated the physiological and behavioural parameters of stress response in laying hens, showing that increased concentrations of plasma corticosterone in response to stress is not directly associated with high levels of fear.

The influence of training-induced sarcomerogenesis on the history dependence of force

Chen, J. — 2020-04-01

The increase or decrease in isometric force following active muscle lengthening or shortening, relative to a reference isometric contraction at the same muscle length and level of activation, are referred to as residual force enhancement (rFE) and residual force depression (rFD), respectively. The purpose of these experiments was to gain further mechanistic insight into the trainability of rFE and rFD, on the basis of serial sarcomere number (SSN) alterations to length-dependent properties. Maximal rFE/rFD measures from the soleus and extensor digitorum longus (EDL) of rats were compared after 4 weeks of uphill/downhill running and a no running control. Serial sarcomere numbers adapted to the training: soleus serial sarcomere number was greater with downhill compared to uphill running, while EDL demonstrated a trend towards more serial sarcomeres for downhill compared to no running. In contrast, absolute and normalized rFE/rFD did not differ across training groups for either muscle. As such, it appears that training-induced SSN adaptations do not modify rFE/rFD at the whole-muscle level. Summary StatementThe addition and subtraction of serial sarcomeres induced by downhill and uphill running, respectively, did not influence the magnitude of stretch-induced force enhancement and shortening-induced force depression.

Melatonin alters fluid phase co-existence in POPC/DPPC/cholesterol membranes

Mei, N. — 2020-04-04

The structure and biophysical properties of lipid biomembranes are important for normal function of plasma and organelle membranes, which is essential for proper functioning of living cells. In Alzheimers disease (AD) the structure of neuronal membranes becomes compromised by the toxic effect of amyloid-{beta} (A{beta}) protein which accumulates at neuron synapses, resulting in membrane perforation and dysfunction, oxidative stress and cell death. Melatonin is an important pineal gland hormone that has been shown to be protective against A{beta} toxicity in cellular and animal studies, but the molecular mechanism of this protection is not well understood. It has been shown that melatonin can interact with model lipid membranes and alter the membrane biophysical properties, such as membrane molecular order and dynamics. This effect of melatonin has been previously studied in simple model bilayers with one or two lipid components, we consider a more complex ternary lipid mixture as our membrane model. In this study, we used 2H-NMR to investigate the effect of melatonin on lipid phase behaviour of a three-component model lipid membranes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol. We used deuterium labelled palmitoyl-d31 in POPC-d31 and DPPC-d62 separately, to probe the changes in hydrocarbon chain order as a function of temperature and varying concentrations of melatonin. We found that melatonin concentration influences phase separation in these ternary mixtures somewhat differently depending on whether POPC-d31 or DPPC-d62was used. At 5 mol% melatonin we observed phase separation in samples with POPC-d31, but not with DPPC-d62. However, at 10 mol% melatonin phase separation was observed in both samples with either POPC-d31 or DPPC-d62. These results indicate that melatonin can have a strong effect on membrane structure and physical properties, which may provide some clues to understanding how melatonin protects against A{beta}. SIGNIFICANCEMelatonin has been shown to be protective against A{beta} pathology in animal and cellular studies. Although the mechanism of this protection is not well-understood, melatonins membrane-active properties may be important in this regard. In this work solid-state deuterium nuclear magnetic resonance was used to study the effect of melatonin on the POPC/DPPC/cholesterol model membranes. Specifically, we showed that melatonin modifies lipid hydrocarbon chain order to promote phase separation. This knowledge helps to explain the role of melatonin in lipid domain formation and may provide a deeper understanding of the mechanism of melatonin neuroprotection in AD.

RubyACRs, non-algal anion channelrhodopsins with highly red-shifted absorption

Govorunova, E. G. — 2020-04-16

Channelrhodopsins are light-gated ion channels widely used to control neuronal firing with light (optogenetics). We report two previously unknown families of anion channelrhodopsins (ACRs), one from the heterotrophic protists labyrinthulomycetes and the other from haptophyte algae. Four closely related labyrinthulomycete ACRs, named RubyACRs here, exhibit a unique retinal binding pocket that creates spectral sensitivities with maxima at 590-610 nm, the most red-shifted channelrhodopsins known, long-sought for optogenetics, and more broadly the most red-shifted microbial rhodopsins so far reported. We identified three spectral tuning residues critical for the red-shifted absorption. Photocurrents recorded from the RubyACR from Aurantiochytrium limacinum (designated AlACR1) under single-turnover excitation exhibited biphasic decay, the rate of which was only weakly voltage-dependent, in contrast to that in previously characterized cryptophyte ACRs, indicating differences in channel gating mechanisms between the two ACR families. Moreover, in A. limacinum we identified three ACRs with absorption maxima at 485, 545, and 590 nm, indicating color-sensitive photosensing with blue, green and red spectral variation of ACRs within individual species of the labyrinthulomycete family. We also report energy transfer from a cytoplasmic fluorescent protein domain to the retinal chromophore bound within RubyACRs, not seen in similar constructs in other channelrhodopsins. Significance StatementOur identification and characterization of two ACR families, one from non-photosynthetic microorganisms, shows that light-gated anion conductance is more widely spread among eukaryotic lineages than previously thought. The uniquely far red-shifted absorption spectra of the subset we designate RubyACRs provide the long-sought inhibitory optogenetic tools producing large passive currents activated by long-wavelength light, enabling deep tissue penetration. Previously only low-efficiency ion-pumping rhodopsins were available for neural inhibition by the orange-red region of the spectrum. The unusual amino acid composition of the retinal-binding pocket in RubyACRs expands our understanding of color tuning in retinylidene proteins. Finally, energy transfer from the fluorescent protein used as a tag on RubyACRs opens a potential new dimension in molecular engineering of optogenetic tools.

Ecosystem Entanglement and the Propagation of Nutrient-Driven Instability

McCann, K. — 2020-04-20

Almost 50 years ago, Michael Rosenzweig pointed out that nutrient addition can destabilize food webs, leading to loss of species and reduced ecosystem function through the paradox of enrichment. Around the same time, David Tilman demonstrated that increased nutrient loading would also be expected to cause competitive exclusion leading to deleterious changes in food web diversity. While both concepts have greatly illuminated general diversity-stability theory, we currently lack a coherent framework to predict how nutrients influence food web stability across a landscape. This is a vitally important gap in our understanding, given mounting evidence of serious ecological disruption arising from anthropogenic displacement of resources and organisms. Here, we combine contemporary theory on food webs and meta-ecosystems to show that nutrient additions are indeed expected to drive loss in stability and function in human-impacted regions. However, this loss in stability occurs not just from wild oscillations in population abundance, but more frequently from the complete loss of an equilibrium due to edible plant species being competitively excluded. In highly modified landscapes, spatial nutrient transport theory suggests that such instabilities can be amplified over vast distances from the sites of nutrient addition. Consistent with this theoretical synthesis, the empirical frequency of these distant propagating ecosystem imbalances appears to be growing. This synthesis of theory and empirical data suggests that human modification of the Earths ecological connectivity is "entangling" once distantly separated ecosystems, causing rapid, expansive, and costly nutrient-driven instabilities over vast areas of the planet. The corollary to this spatial nutrient theory, though -- akin to weak interaction theory from food web networks -- is that slow spatial nutrient pathways can be potent stabilizers by moderating flows across a landscape.

Investigating the effects of incremental conditioning and supplemental dietary tryptophan on the voluntary activity and behaviour of mid-distance training sled dogs

Robinson, E. — 2020-04-21

Serotonin is a neurotransmitter synthesized by the amino acid tryptophan (Trp) that has the potential to impact the behaviour and activity of dogs. The objective of this study was to assess the effects of supplemental Trp and a 12-week incremental training regimen on the voluntary activity and behaviour of client-owned Siberian Huskies. Sixteen dogs were blocked for age, BW and sex and then randomly allocated to either the control or treatment group. Both groups were fed the same dry extruded diet; however, the treatment group were supplemented with Trp to achieve a Trp: large neutral amino acid ratio of 0.075:1. Once a week, a 5-minute video recording was taken immediately pre- and post- exercise to evaluate dogs behaviours. Activity monitors were used to record voluntary activity on both training and rest days. Linear regression analysis was used to assess the relationship between training week and time spent performing each behaviour. Additionally, a repeated measure mixed model was used to test differences between diet groups and training week for both behavioural and activity count data. The time spent performing agonistic behaviours prior to exercise was negatively associated with week for treatment dogs ({beta} = -0.32, 95% CI [-0.55, -0.10], P < 0.05) and no change was observed for control dogs ({beta} = -0.13, 95% CI [-0.41, 0.15], P > 0.10). Treatment did not have any effect on activity levels (P > 0.10). For all dogs, locomotive behaviours decreased prior to exercise as weeks progressed (P < 0.05), while run day voluntary activity depended on the distance run that day (P < 0.05). These data suggest that sled dogs experience an exercise-induced reduction in voluntary locomotion in response to both single bouts and repetitive bouts of exercise. Additionally, tryptophan supplementation may decrease agonistic behaviours, without having any effect on voluntary activity.

Massive Multiplexing Can Deliver a $1 Test for COVID-19

Hebert, P. D. — 2020-05-08

The severe acute respiratory syndrome virus, SARS-CoV-2 (hereafter COVID-19), rapidly achieved global pandemic status, provoking large-scale screening programs in many nations. Their activation makes it imperative to identify methods that can deliver a diagnostic result at low cost. This paper describes an approach which employs sequence variation in the gene coding for its envelope protein as the basis for a scalable, inexpensive test for COVID-19. It achieves this by coupling a simple RNA extraction protocol with low-volume RT-PCR, followed by E-Gel screening and sequencing on high-throughput platforms to analyze 10,000 samples in a run. Slight modifications to the protocol could support screening programs for other known viruses and for viral discovery. Just as the $1,000 genome is transforming medicine, a $1 diagnostic test for viral and bacterial pathogens would represent a major advance for public health.

CommDivMap: Modelling and mapping species richness at different spatial scales

Miller, J. E. — 2020-05-13

1. Modern ecosystem models have the potential to greatly enhance our capacity to predict community responses to change, but they demand comprehensive spatial distribution information, creating the need for new approaches to gather and synthesize biodiversity data. 2. Metabarcoding or metagenomics can generate comprehensive biodiversity data sets at species-level resolution but they are limited to point samples. 3. CommDivMap contains a number of functions that can be used to turn OTU tables resulting from metabarcoding runs of bulk samples into species richness maps. We tested the method on a series of arthropod bulk samples obtained from various experimental agricultural plots. 4. The script runs smoothly and is reasonably fast. We hope that our assemble first, predict later approach to statistical modelling of species richness will set the stage for the transition from data-rich but finite sets of point samples to spatially continuous biodiversity maps.

Effects of parity, blood progesterone, and non-steroidal anti-inflammatory treatment on the dynamics of the uterine microbiota of healthy postpartum dairy cows

Bogado Pascottini, O. — 2020-05-18

This study evaluated the effects of treatment with meloxicam (a non-steroidal anti-inflammatory drug), parity, and blood progesterone concentration on the dynamics of the uterine microbiome of clinically healthy postpartum dairy cows. Seven primiparous and 9 multiparous postpartum Holstein cows received meloxicam (0.5 mg/kg SC, n = 7 cows) once daily for 4 days (10 to 13 days in milk (DIM)) or were untreated (n = 9 cows). Endometrial cytology samples were collected by cytobrush at 10, 21, and 35 DIM, from which the metagenomic analysis was done using 16S rRNA gene sequence analysis. A radioimmunoassay was used to measure progesterone concentration in blood serum samples at 35 DIM and cows were classified as > 1 ng/mL (n = 10) or [≤] 1 ng/mL (n = 6). Alpha diversity for bacterial genera (Chao1, Shannon-Weiner, and Camargos evenness indices) were not affected by DIM, meloxicam treatment, parity, or progesterone category (P > 0.2). For beta diversity (genera level), principal coordinate analysis (Bray-Curtis) showed differences in microbiome between parity groups (P = 0.01). There was lower overall abundance of Anaerococcus, Bifidobacterium, Corynebacterium, Lactobacillus, Paracoccus, Staphylococcus, and Streptococcus and higher abundance of Bacillus, Fusobacterium, and Novosphingobium in primiparous than multiparous cows (P < 0.05); these patterns were consistent across sampling days. Bray-Curtis dissimilarity did not differ by DIM at sampling, meloxicam treatment, or progesterone category at 35 DIM (P > 0.5). In conclusion, uterine bacterial composition was not different at 10, 21, or 35 DIM, and meloxicam treatment or progesterone category did not affect uterine microbiota in clinically healthy postpartum dairy cows. Primiparous cows presented a different composition of uterine bacteria than multiparous cows. The differences in microbiome associated with parity might be attributable to changes that occur consequent to the first calving, but this hypothesis should be investigated further.

A2G2: A Python wrapper to perform very largealignments in semi-conserved regions

Hleap, J. S. — 2020-05-26

SummaryAmplicons to Global Gene (A2G2) is a Python wrapper that uses MAFFT and an "Amplicon to Gene" strategy to align very large numbers of sequences while improving alignment accuracy. It is specially developed to deal with conserved genes, where traditional aligners introduce a significant amount of gaps. A2G2 leverages the add sequences option of MAFFT to align the sequences to a global reference gene and a local reference region. Both of these references can be consensus sequences of trusted sources. Efficient parallelization of these tasks allows A2G2 to align a very large number of sequences (> 500K) in a reasonable amount of time. A2G2 can be imported in Python for easier integration with other software, or can be run via command line. AvailabilityA2G2 is implemented in Python 3 (3.6) and depends on MAFFT availability. Other package requirements can be found in the requirements.txt file at https://github.com/jshleap/A2G. A2G2 is also available via PyPi (https://pypi.org/project/A2G). It is licensed under the LGPLv3. Supplementary informationSupplementary material is available at github as jupyter notebook.

Moyamoya Disease-Associated RNF213 Alleles Encode Dominant Negative Alleles That Globally Impair Ubiquitylation

Bhardwaj, A. — 2020-05-26

Single nucleotide polymorphisms (SNPs) in RNF213, which encodes a 591kDa protein with AAA+ ATPase and RING E3 domains, are associated with a rare, autosomal dominant cerebrovascular disorder, Moyamoya disease (MMD). MMD-associated SNPs primarily localize to the C-terminal region of RNF213, and some affect conserved residues in the RING domain. Although the autosomal dominant inheritance of MMD could most easily be explained by RNF213 gain-of-function, the type of ubiquitylation catalyzed by RNF213 and the effects of MMD-associated SNPs on its E3-ligase activity have remained unclear. We found that the RING domain of RNF213 uses the E2-conjugating enzyme UBE2D2 to catalyze predominantly K6-dependent poly-ubiquitination events comprising a mixture of typical and atypical ubiquitin linkages. MMD-associated SNPs encode proteins with decreased E3-ligase activity and the most frequent MMD allele, RNF213R4810K, is a dominant negative mutant that decreases ubiquitylation globally. By contrast, MMD-associated RNF213 SNPs do not affect ATPase activity. We propose that decreased RNF213 E3-ligase activity is central to MMD pathogenesis.

Freshwater diatom biomonitoring through benthic kick-net metabarcoding

Maitland, V. C. — 2020-05-28

Biomonitoring is an essential tool for assessing ecological conditions and informing management strategies. The application of DNA metabarcoding and high throughput sequencing has improved data quantity and resolution for biomonitoring of taxa such as macroinvertebrates, yet, there remains the need to optimise these methods for other taxonomic groups. Diatoms have a longstanding history in freshwater biomonitoring as bioindicators of water quality status. However, periphyton scraping, a common diatom sampling practice, is time-consuming and thus costly in terms of labour. This study examined whether the benthic kick-net technique used for macroinvertebrate biomonitoring could be applied to bulk-sample diatoms for metabarcoding. To test this approach, we collected samples using both conventional microhabitat periphyton scraping and bulk-tissue kick-net methodologies in parallel from replicated sites with different habitat status (good/fair). We found there was no significant difference in community assemblages between conventional periphyton scraping and kick-net methodologies, but there was significant difference between diatom communities depending on site quality (P = 0.029). These results show the diatom taxonomic coverage achieved through DNA metabarcoding of kick-net is suitable for ecological biomonitoring applications. The shift to a more robust sampling approach and capturing diatoms and macroinvertebrates in a single sampling event has the potential to significantly improve efficiency of biomonitoring programmes.

bbsBayes: An R Package for Hierarchical Bayesian Analysis of North American Breeding Bird Survey Data

Edwards, B. P. — 2020-05-30

The North American Breeding Bird Survey (BBS) is the primary ecological monitoring program used to assess the population, status, and trend of North American birds. As such, accessible analysis of BBS data is crucial to wildlife conservation/management and ecological science in North America. The R package bbsBayes was developed as a wrapper for the analysis of BBS data using hierarchical Bayesian models, including the models currently used by the Canadian Wildlife Service and the United States Geological Survey. The goal of bbsBayes is to provide an accessible package for anyone in the conservation community to estimate population trajectories (time-series) and trends (rates of change) for any of the 400+ bird species monitored by the BBS, and to allow more advance users to easily access the data and model-templates necessary to customize an analysis for their research.

Early temporal dynamics of cellular responses to SARS-CoV-2

Banerjee, A. — 2020-06-18

Type I interferons (IFNs) are our first line of defence against a virus. Protein over-expression studies have suggested the ability of SARS-CoV-2 proteins to block IFN responses. Emerging data also suggest that timing and extent of IFN production is associated with manifestation of COVID-19 severity. In spite of progress in understanding how SARS-CoV-2 activates antiviral responses, mechanistic studies into wildtype SARS-CoV-2-mediated induction and inhibition of human type I IFN responses are lacking. Here we demonstrate that SARS-CoV-2 infection induces a mild type I IFN response in vitro and in moderate cases of COVID-19. In vitro stimulation of type I IFN expression and signaling in human airway epithelial cells is associated with activation of canonical transcriptions factors, and SARS-CoV-2 is unable to inhibit exogenous induction of these responses. Our data demonstrate that SARS-CoV-2 is not adept in blocking type I IFN responses and provide support for ongoing IFN clinical trials. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/158154v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@193c540org.highwire.dtl.DTLVardef@7b106forg.highwire.dtl.DTLVardef@1741cfforg.highwire.dtl.DTLVardef@1bde68_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL SUMMARY C_FIG

Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study

Monthony, A. S. — 2020-06-24

Cannabis sativa is relatively recalcitrant to regeneration from somatic tissues, but several reports have been published demonstrating a response. Most reports show low levels of regeneration from somatic tissues, but a landmark publication by Lata et al. in 2010 reported regeneration from leaf explants with a 96% response rate, producing an average of 12.3 shoots per explant in a single, high-THC genotype. Despite the importance regeneration plays in plant biotechnology this protocol has not been used in subsequent papers in the decade since it was published, raising the concern that it is not reproducible. Many researchers are looking to build research programmes in this growing field, and it is important that the reproducibility and robustness of single-genotype C. sativa regeneration protocols undergo multi-lab validations to ensure they are reproducible across the species. Replication studies in this burgeoning field will help research groups avoid lost time and resources which arise from pursuing protocols that are not reproducible. Here we test the replicability of this protocol across 10 drug-type C. sativa genotypes. This protocol successfully induced callus in all 10 genotypes. Callus size and appearance substantially differed among cultivars, with the most responsive genotype producing 6-fold more callus than the least responsive genotype. However, the most successful shoot induction medium developed in the 2010 paper failed to induce regeneration in any of the cultivars tested, resulting in the eventual necrosis of the calli. Based on this replication study, it is evident that the existing regeneration protocol is not robust and could not be replicated in any of the 10 genotypes tested.View Full Text

Alignment-free identification of COI DNA barcode data with the Python package Alfie

Nugent, C. M. — 2020-06-29

Characterization of biodiversity from environmental DNA samples and bulk metabarcoding data is hampered by off-target sequences that can confound conclusions about a taxonomic group of interest. Existing methods for isolation of target sequences rely on alignment to existing reference barcodes, but this can bias results against novel genetic variants. Effectively parsing targeted DNA barcode data from off-target noise improves the quality of biodiversity estimates and biological conclusions by limiting subsequent analyses to a relevant subset of available data. Here, we present Alfie, a Python package for the alignment-free classification of cytochrome c oxidase subunit I (COI) DNA barcode sequences to taxonomic kingdoms. The package determines k-mer frequencies of DNA sequences, and the frequencies serve as input for a neural network classifier that was trained and tested using ~58,000 publicly available COI sequences. The classifier was designed and optimized through a series of tests that allowed for the optimal set of DNA k-mer features and optimal machine learning algorithm to be selected. The neural network classifier rapidly assigns COI sequences to kingdoms with greater than 99% accuracy and is shown to generalize effectively and make accurate predictions about data from previously unseen taxonomic classes. The package contains an application programming interface that allows the Alfie packages functionality to be extended to different DNA sequence classification tasks to suit a users need, including classification of different genes and barcodes, and classification to different taxonomic levels. Alfie is free and publicly available through GitHub (https://github.com/CNuge/alfie) and the Python package index (https://pypi.org/project/alfie/).

Identification of antimicrobial resistance-associated genes through whole genome sequencing of high and low MIC isolates of Mycoplasma bovis

Ledger, L. — 2020-07-01

Antimicrobial resistance (AMR) in Mycoplasma bovis has previously been associated with mutations within topoisomerases and ribosomal genes rather than specific resistance-conferring genes. This study used whole genome sequencing (WGS) to identify potential new AMR mechanisms. It was found that a 2019 clinical isolate with high MIC (2019-043682) had a new core genome multilocus sequencing (cgMLST) type (ST10-like) and 91% homology with the published genome of M. bovis PG45. Closely related to PG45, a 1982 isolate (1982-M6152) shared the same cgMLST type (ST17), 97.2% homology and similar low MIC results. Known and potential AMR-associated genetic events were identified through comparison with the published M. bovis PG45 genome. Isolate 2019-043682 had 507 genes with non-synonymous mutations (NSMs) and 67 genes disrupted. Isolate 1982-M6152 had 81 NSMs and 20 disruptions. Based on functional roles and known mechanisms of antimicrobials, a 55 gene subset was assessed for potential AMR mechanisms. Of these, 14 were previously identified from other bacteria as sites of AMR mutation, 41 shared similar functions to them, and 11 contained gene-disrupting mutations. This study indicated that M. bovis may obtain high AMR characteristics by mutating or disrupting other functional genes, in addition to topoisomerases and ribosomal genes.

METAWORKS: A flexible, scalable bioinformatic pipeline for multi-marker biodiversity assessments

Porter, T. M. — 2020-07-15

Multi-marker metabarcoding is increasingly being used to generate biodiversity information across different domains of life from microbes to fungi to animals such as for molecular ecology and biomonitoring applications in different sectors from academic research to regulatory agencies and industry. Current popular bioinformatic pipelines support microbial and fungal marker analysis, while ad hoc methods are often used to process animal metabarcode markers from the same study. MetaWorks provides a harmonized processing environment, pipeline, and taxonomic assignment approach for demultiplexed Illumina reads for all biota using a wide range of metabarcoding markers such as 16S, ITS, and COI. A Conda environment is provided to quickly gather most of the programs and dependencies for the pipeline. Several workflows are provided such as: taxonomically assigning exact sequence variants, provides an option to generate operational taxonomic units, and facilitates single-read processing. Pipelines are automated using Snakemake to minimize user intervention and facilitate scalability. All pipelines use the RDP classifier to provide taxonomic assignments with confidence measures. We extend the functionality of the RDP classifier for taxonomically assigning 16S (bacteria), ITS (fungi), and 28S (fungi), to also support COI (animals), rbcL (eukaryotes, land plants, diatoms), 12S (fish), 18S (eukaryotes, diatoms) and ITS (fungi, plants). MetaWorks properly handles ITS by trimming flanking conserved rRNA gene regions as well as protein coding genes by providing two options for removing obvious pseudogenes. MetaWorks is available at https://github.com/terrimporter/MetaWorks along with quick-start instructions using test data, detailed workflow descriptions, and a tutorial for new users.

Deconstructing biodiversity-ecosystem function relationships: Filtering of macroinvertebrate traits in a large river floodplain

Rideout, N. K. — 2020-07-15

The Biodiversity-Ecosystem Function hypothesis postulates that higher biodiversity is correlated with ecosystem function by providing a high number of filled niches through species response types and resource use patterns. Through their high spatio-temporal habitat diversity, floodplains are highly productive ecosystems, supporting communities that are naturally resilient and highly diverse. We examined linkages among floodplain wetland habitats, invertebrate communities and their associated traits, and ecosystem function across 60 sites within the floodplain wetlands of the lower Wolastoq | Saint John River, New Brunswick, using structural equation modelling and Threshold Indicator Taxa ANalysis (TITAN2). We identified key environmental filters of invertebrate communities, namely linking increased niche differentiation through historical change, flood pulse dynamics, and macrophyte bed complexity with increased taxa and functional diversity. Examination of traits linked to ecosystem functions revealed that healthy wetlands with higher primary productivity were associated with greater functional evenness and richness, while habitat patches with increased decomposition rates had low functional richness, reflecting highly disturbed habitat. Our results highlight key differences between wetland and riverine ecosystems, relating to how critical functions support healthy wetland habitats by providing increased resilience to disturbance, here associated with differing levels of conservation protection.

Assessment of current taxonomic assignment strategies for metabarcoding eukaryotes

Hleap, J. S. — 2020-07-22

The effective use of metabarcoding in biodiversity science has brought important analytical challenges due to the need to generate accurate taxonomic assignments. The assignment of sequences to a generic or species level is critical for biodiversity surveys and biomonitoring, but it is particularly challenging. Researchers must select the approach that best recovers information on species composition. This study evaluates the performance and accuracy of seven methods in recovering the species composition of mock communities which vary in species number and specimen abundance, while holding upstream molecular and bioinformatic variables constant. It also evaluates the impact of parameter optimization on the quality of the predictions. Despite the general belief that BLAST top hit underperforms newer methods, our results indicate that it competes well with more complex approaches if optimized for the mock community under study. For example, the two machine learning methods that were benchmarked proved more sensitive to the reference database heterogeneity and completeness than methods based on sequence similarity. The accuracy of assignments was impacted by both species and specimen counts which will influence the selection of appropriate software. We urge the usage of realistic mock communities to allow optimization of parameters, regardless of the taxonomic assignment method used.

North American deer mice are susceptible to SARS-CoV-2

Griffin, B. D. — 2020-07-26

The zoonotic spillover of the pandemic SARS-coronavirus 2 (SARS-CoV-2) from an animal reservoir, currently presumed to be the Chinese horseshoe bat, into a naive human population has rapidly resulted in a significant global public health emergency. Worldwide circulation of SARS-CoV-2 in humans raises the theoretical risk of reverse zoonosis events with wildlife, reintroductions of SARS-CoV-2 into permissive non-domesticated animals, potentially seeding new host reservoir species and geographic regions in which bat SARS-like coronaviruses have not historically been endemic. Here we report that North American deer mice (Peromyscus maniculatus) and some closely related members of the Cricetidae family of rodents possess key amino acid residues within the angiotensin-converting enzyme 2 (ACE2) receptor known to confer SARS-CoV-2 spike protein binding. Peromyscus rodent species are widely distributed across North America and are the primary host reservoirs of several emerging pathogens that repeatedly spill over into humans including Borrelia burgdorferi, the causative agent of Lyme disease, deer tick virus, and Sin Nombre orthohantavirus, the causative agent of hantavirus pulmonary syndrome (HPS). We demonstrate that adult deer mice are susceptible to SARS-CoV-2 infection following intranasal exposure to a human isolate, resulting in viral replication in the upper and lower respiratory tract with little or no signs of disease. Further, shed infectious virus is detectable in nasal washes, oropharyngeal and rectal swabs, and viral RNA is detectable in feces and occasionally urine. We further show that deer mice are capable of transmitting SARS-CoV-2 to naive deer mice through direct contact. The extent to which these observations may translate to wild deer mouse populations remains unclear, and the risk of reverse zoonosis and/or the potential for the establishment of Peromyscus rodents as a North American reservoir for SARS-CoV-2 is unknown. Nevertheless, efforts to monitor wild, peri-domestic Peromyscus rodent populations are likely warranted as the SARS-CoV-2 pandemic progresses.

Model-based genotype and ancestry estimation for potential hybrids with mixed-ploidy

Shastry, V. — 2020-08-03

Non-random mating among individuals can lead to spatial clustering of genetically similar individuals and population stratification. This deviation from panmixia is commonly observed in natural populations. Consequently, individuals can have parentage in single populations or involving hybridization between differentiated populations. Accounting for this mixture and structure is important when mapping the genetics of traits and learning about the formative evolutionary processes that shape genetic variation among individuals and populations. Stratified genetic relatedness among individuals is commonly quantified using estimates of ancestry that are derived from a statistical model. Development of these models for polyploid and mixed-ploidy individuals and populations has lagged behind those for diploids. Here, we extend and test a hierarchical Bayesian model, called entropy, which can use low-depth sequence data to estimate genotype and ancestry parameters in autopolyploid and mixed-ploidy individuals (including sex chromosomes and autosomes within individuals). Our analysis of simulated data illustrated the trade-off between sequencing depth and genome coverage and found lower error associated with low depth sequencing across a larger fraction of the genome than with high depth sequencing across a smaller fraction of the genome. The model has high accuracy and sensitivity as verified with simulated data and through analysis of admixture among populations of diploid and tetraploid Arabidopsis arenosa.

Metabarcoding, direct stomach observation and stable isotope analysis reveal a highly diverse diet for the invasive green crab in Atlantic Patagonia

Cordone, G. — 2020-08-14

The European green crab Carcinus maenas and its sister species C. aestuarii are highly invasive species causing damage to coastal ecosystems and contributing to severe economic losses worldwide. C. maenas was first detected at the Atlantic Patagonian coast in 2001. In this work, we studied the diet of the green crab in a recently invaded location in Golfo Nuevo, using three complementary techniques: direct stomach observation, metabarcoding of gut content and stable isotope analysis. Direct stomach observation and metabarcoding showed that green crabs have a broad omnivorous diet, ingesting most of the phyla present in the study area. Gut content metabarcoding allowed a detailed description of algal diversity and revealed other taxa that went unnoticed in the visual stomach analysis. Stable isotope analysis showed that the major contribution to the crabs diet was from the phytoplankton chain (by bivalve consumption) and not directly from algae. This study approach combining three complementary techniques also allowed us to detect some differences in the diet between sexes, which suggests that male and female crabs are not as ecologically equivalent as previously thought. Besides, we detected sequences corresponding to C. aestuarii suggesting that the green crab Patagonian population is a hybrid of both sister species. These findings are key to understanding the impacts green crabs can have on the local ecosystem.

Comparison of methods for milk pre-processing, exosome isolation, and RNA extraction in bovine and human milk

Wijenayake, S. — 2020-08-14

Milk is a highly complex, heterogeneous biological fluid that contains bioactive, membrane-bound extracellular vesicles called exosomes. Characterization of milk-derived exosomes (MDEs) is challenging due to the lack of standardized methods that are currently being used for milk pre-processing, exosome isolation, and RNA extraction. In this study, we tested: 1) three pre-processing methods to remove cream, fat, and casein proteins from bovine milk to determine whether pre-processing of whole milk, prior to long-term storage, improves MDE isolations, 2) two commonly-used exosome isolation methods, and 3) four extraction protocols for obtaining high quality MDE RNA from bovine and human milk. MDEs were characterized via Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). We also present an optimized method of TEM sample preparation and isolation of total soluble protein from MDEs. Our results indicated that: 1) pre-processing of bovine milk prior to storage does not affect the final exosome yield or the purity, 2) ExoQuick precipitation is better suited for MDE isolation than ultracentrifugation for bovine and human milk, and 3) TRIzol LS produced the highest RNA yield in bovine milk, whereas TRIzol LS, TRIzol+RNA Clean and Concentrator, and TRIzol LS+RNA Clean and Concentrator methods can be used for human milk.

Which actin genes are necessary for zebrafish heart development and function?

Prill, K. — 2020-08-26

Heart failure is the number one cause of mortality in the world, contributed to by cardiovascular disease. Many diseases of the heart muscle are caused by mutations in genes encoding contractile proteins, including cardiac actin mutations. Zebrafish are an advantageous system for modeling cardiac disease since embryos can develop without a functional heart. However, genome duplication in the teleost lineage creates a unique obstacle by increasing the number of genes involved in heart development. Four actin genes are expressed in the zebrafish heart: acta1b; actc1c; and duplicates of actc1a on chromosome 19 and 20. Here, we characterize the actin genes involved in early zebrafish heart development using in situ hybridization and CRISPR targeting to determine which gene is best to model changes seen in human patients with heart disease. The actc1a and acta1b genes are predominant during embryonic heart development, resulting in severe cardiac phenotypes when targeted with CRISPRs. Targeting these two cardiac genes with CRISPRs simultaneously results in a more severe phenotype than their individual counterparts, with the results suggesting compensation for lost actin genes by other actin paralogues. Given the duplication of the actc1a gene, we recommend acta1b as the best gene for targeted cardiac actin research.

A generalist lifestyle allows rare Gardnerella spp. to persist at low levels in the vaginal microbiome

Khan, S. — 2020-08-25

Gardnerella spp. are considered a hallmark of bacterial vaginosis, a dysbiosis of the vaginal microbiome. There are four cpn60 sequence-based subgroups within the genus (A, B, C, and D), and thirteen genome species have been defined recently. Gardnerella spp. co-occur in the vaginal microbiome with varying abundance, and these patterns are shaped by a resource-dependent, exploitative competition, which affects the growth rate of subgroup A, B, and C negatively. The growth rate of rarely abundant subgroup D, however, increases with the increasing number of competitors, negatively affecting the growth rate of others. We hypothesized that a nutritional generalist lifestyle and minimal niche overlap with the other, more abundant Gardnerella spp. facilitate the maintenance of subgroup D in the vaginal microbiome through negative-frequency dependent selection. Using 40 whole genome sequences from isolates representing all four subgroups we found that they could be distinguished based on content of their predicted proteomes. Proteins associated with carbohydrate and amino acid uptake and metabolism were significant contributors to the separation of subgroups. Subgroup D isolates had significantly more of their proteins assigned to amino acid metabolism than the other subgroups. Subgroup D isolates were also significantly different from others in terms of number and type of carbon sources utilized in a phenotypic assay, while the other three could not be distinguished. Overall, the results suggest that a generalist lifestyle and lack of niche overlap with other Gardnerella spp. leads to subgroup D being favoured by negative-frequency dependent selection in the vaginal microbiome.

The novel roles of choline transporter-like 1 and 2 in ethanolamine transport

Taylor, A. — 2020-08-28

We examined a novel function of mammalian Choline-Transporter-Like proteins CTL1/SLC44A1 and CTL2/SLC44A2 in ethanolamine transport. We established two distinct ethanolamine transport systems of a high affinity (K1 = 55.6 - 66.5 M), mediated by CTL1, and of a low affinity (K2 = 275 - 299 M), mediated by CTL2. Both types of transport are Na+-independent and mediated in a pH dependent manner, as expected for ethanolamine/H+ antiporters. Primary human fibroblasts with separate frameshift mutations (M1= SLC44A1 {Delta}Asp517 and M2= SLC44A1 {Delta}Ser126) are devoid of CTL1 ethanolamine transport but maintain unaffected CTL2 transport. The lack of CTL1 or CTL2 reduced the ethanolamine transport, the flux by the CDP-ethanolamine Kennedy pathway and PE synthesis. Overexpression of CTL1 in SLC44A1 {Delta}Ser126 (M2) cells improved the ethanolamine transport and PE synthesis. The SLC44A1 {Delta}Ser126 cells are reliant on CTL2 function and CTL2 siRNA almost completely abolished ethanolamine transport in the whole cells and mitochondria. Overexpression of CTL1 and CTL2 cDNAs increased ethanolamine transport in control and SLC44A1{Delta}Ser126 cells. CTL1 and CTL2 facilitated mitochondrial ethanolamine uptake, but the transport mediated by CTL1 is predominant in the whole cells and mitochondria. These data firmly established that CTL1 and CTL2 are the first identified ethanolamine transporters in the whole cells and mitochondria, with intrinsic roles in de novo PE synthesis by the CDP-Etn Kennedy pathway and compartmentation of intracellular ethanolamine. SignificanceThe lack of Choline Transporter Like 1 (SLC44A1/CTL1) is the primary cause of a new neurodegenerative disorder with elements of childhood-onset parkinsonism and mitochondrial dysfunction. SLC44A2/CTL2 encodes the human neutrophil antigen 3, causes autoimmune hearing loss and Menieres disease, and has been recently identified as the main risk factor for thrombosis-the major cause of death in Covid-19 patients. Our investigation provides insights into the novel functions of CTL1 and CTL2 as intrinsic ethanolamine transporters. CTL1 and CTL2 are high and low affinity transporters, with direct roles in the membrane phospholipid synthesis. The work contributes to new knowledge for CTL1 and CTL2 independent transport functions and the optimization of prevention and treatment strategies in those various diseases.

When conflicts get heated, so does the planet: social-climate dynamics under inequality

Menard, J. — 2020-09-16

Climate dynamics are inextricably linked to processes in social systems that are highly unequal. This suggests a need for coupled social-climate models that capture pervasive real-world asymmetries in the population distribution of the consequences of anthropogenic climate change and climate (in)action. Here we develop a simple social-climate model with group structure to investigate how anthropogenic climate change and population heterogeneity co-evolve. We find that greater homophily and resource inequality cause an increase in the global peak temperature anomaly by as much as 0.7{degrees}C. Also, climate change can structure human populations by driving opinion polarization. Finally, climate mitigation achieved by reducing the cost of mitigation measures paid by individuals tends to be contingent upon socio-economic conditions, whereas policies that achieve communication between different strata of society show climate mitigation benefits across a broad socio-economic regime. We conclude that advancing climate change mitigation efforts can benefit from a social-climate systems perspective.

Coupled social-land dynamics and the future of sustainable consumption

Pal, S. — 2020-09-20

Dietary patterns have long been a driver of global land use. Increasingly, they also respond to it, in part because of social forces that support adoption of sustainable diets. Here we develop a coupled social-land use dynamics model parameterised for 164 countries. We project global land use under 20 scenarios for future population, income, and agricultural yield. When future yields are low and/or population size is high, coupled social-land feedbacks can reduce the peak global land use by up to 2 billion hectares, if socio-economic barriers to adopting a sustainable diet are sufficiently low. In contrast, when population growth is low or yield is high, reductions in income elasticity can increase peak land use by 100 million hectares. The model also exhibits a regime of synergistic effects whereby simultaneous changes to multiple social and economic parameters are required to change land use projections. This research demonstrates the value of including coupled social-land feedbacks in land use projections.

At each site its diversity: DNA barcoding reveals remarkable earthworm diversity in neotropical rainforests of French Guiana

Maggia, M.-E. — 2020-09-23

Despite their recognized essential role in soil, earthworms in tropical environments are still understudied. The aim of this study was to re-evaluate the diversity at the regional scale, as well as to investigate the environmental and spatial drivers of earthworm communities. We sampled earthworm communities across a range of habitats at six locations in French Guiana using three different sampling methods. We generated 1675 DNA barcodes and combined them with data from a previous study. Together, all sequences clustered into 119 MOTUs which were used as proxy to assess species richness. Only two MOTUs were common between the six locations and 20.2 % were singletons, showing very high regional species richness and a high number of rare species. A canonical redundancy analysis was used to identify key drivers of the earthworm community composition. The RDA results and beta-diversity calculations both show strong species turnover and a strong spatial effect, resulting from dispersal limitations that are responsible for the current community composition. Sampling in different microhabitats allowed the discovery of 23 MOTUs that are exclusively found in decaying trunks and epiphytes, highlighting hidden diversity of earthworms outside of soil.

The 4R Genome Duplication In Salmonine Fishes: Insights From Conserved Non-Coding Elements

Castillo, A. H. — 2020-09-25

Gene and genome duplications are essential processes in evolution. Salmonids are ideal animal model systems to study these processes, as they originated from a tetraploid ancestor. Conserved non-coding elements (CNEs) are of interest because of their highly conserved DNA consensus motifs spanning lineages as diverse and divergent as humans and fish. The main goal of this study is to test CNEs as a tool to study genome duplications and to revisit the "4R" hypothesis and phylogeny of Salmonine fishes (Salmonidae) Salmo salar, Salvelinus alpinus and Oncorhynchus mykiss through the study of copy number and nucleotide variation in six pairs of CNEs. Allele numbers for most CNE sequence pairs are consistent with the 4R hypothesis, as is the symmetric phylogenetic topology shown by some CNE pairs; the estimated date of CNE duplication is consistent with the only reported range of 25-100Mya. However, the phylogenetic relationships within Salmoninae remain unresolved.

Effects of Malaise trap spacing on species richness and composition of terrestrial arthropod bulk samples

Steinke, D. — 2020-10-02

The Malaise trap has gained popularity for assessing diverse terrestrial arthropod communities because it collects large samples with modest effort. A number of factors that influence collection efficiency, placement being one of them. For instance, when designing larger biotic surveys using arrays of Malaise traps we need to know the optimal distance between individual traps that maximises observable species richness and community composition. We examined the influence of spacing between Malaise traps by metabarcoding samples from two field experiments at a site in Waterloo, Ontario, Canada. For one experiment, we used two trap pairs deployed at weekly increasing distance (3m increments from 3 to 27 m). The second experiment involved a total of 10 traps set up in a row at 3m distance intervals for three consecutive weeks. Results show that community similarity of samples decreases over distance between traps. The amount of species shared between trap pairs shows drops considerably at about 15m trap-to-trap distance. This change can be observed across all major taxonomic groups and for two different habitat types (grassland and forest). Large numbers of OTUs found only once within samples cause rather large dissimilarity between distance pairs even at close proximity. This could be caused by a large number of transient species from adjacent habitat which arrive at the trap through passive transport, as well as capture of rare taxa, which end up in different traps by chance.

Spatial fingerprinting: horizontal fusion of multi-dimensional bio-tracers as solution to global food provenance problems

Cazelles, K. — 2020-10-03

Building the capacity of efficiently determining the provenance of food products represents a crucial step towards the sustainability of the global food system. Whether it is for enforcing existing egislation or providing reliable information to consumers, technologies to verify geographical origin of food are being actively developed. Biological tracers (bio-tracers) such as DNA and stable isotopes have recently demonstrated their potential for determining provenance. Here we show that the data fusion of bio-tracers is a very powerful technique for geographical provenance discrimination. Based on 90 individuals of Sockeye salmon that originate from 3 different areas for which we measured 17 bio-tracers, we demonstrate that increasing the combined bio-tracers results in stronger the discriminatory power. The generality of our results are mathematically demonstrated under simplifying assumptions and numerically confirmed in our case study using three commonly used supervised learning techniques.

Neonicotinoid exposure affects foraging, nesting, and reproductive success of ground-nesting solitary bees

Willis Chan, D. S. — 2020-10-08

Despite their indispensable role in food production1,2, insect pollinators are threatened by multiple environmental stressors, including pesticide exposure2-4. Although honeybees are important, most pollinating insect species are wild, solitary, ground-nesting bees1,4-6 that are inadequately represented by honeybee-centric regulatory pesticide risk assessment frameworks7,8. Here, for the first time, we evaluate the effects of realistic exposure to systemic insecticides (imidacloprid, thiamethoxam or chlorantraniliprole) on a ground-nesting bee species in a semi-field experiment. Hoary squash bees (Eucera (Peponapis) pruinosa) provide essential pollination services to North American pumpkin and squash crops9-14 and commonly nest within cropping areas10, placing them at risk of exposure to pesticides in soil8,10, nectar and pollen15,16. Hoary squash bees exposed to an imidacloprid-treated crop initiated 85% fewer nests, left 84% more pollen unharvested, and produced 89% fewer offspring than untreated controls. We found no measurable impact on squash bees from exposure to thiamethoxam- or chlorantraniliprole-treated crops. Our results demonstrate important sublethal effects of field-realistic exposure to a soil-applied neonicotinoid (imidacloprid) on the behaviour and reproductive success of a ground-nesting solitary bee. To prevent potential declines in ground-nesting bee populations and associated impoverishment of crop pollination services, soil must be considered a possible route of pesticide exposure for bees, and restrictions on soil-applied insecticides may be justified.

In Pursuit of a Better Broiler: Growth, Efficiency and Mortality of 16 Strains of Broiler Chickens

Torrey, S. — 2020-10-16

To meet the growing consumer demand for chicken meat, the poultry industry has selected broiler chickens for increasing efficiency and breast yield. While this high productivity means affordable and consistent product, it has come at a cost to broiler welfare. There has been increasing advocacy and consumer pressure on primary breeders, producers, processors and retailers to improve the welfare of the billions of chickens processed annually. Several small-scale studies have reported better welfare outcomes for slower growing strains compared to fast growing, conventional strains. However, these studies often housed birds with range access or used strains with vastly different growth rates. Additionally, there may be traits other than growth, such as body conformation, that influence welfare. As the global poultry industries consider the implications of using slower growing strains, there was a need for a comprehensive, multidisciplinary examination of broiler chickens with a wide range of genotypes differing in growth rate and other phenotypic traits. To meet this need, our team designed a study to benchmark data on conventional and slower growing strains of broiler chickens reared in standardized laboratory conditions. Over a two-year period, we studied 7,528 broilers from 16 different genetic strains. In this paper, we compare the growth, efficiency and mortality of broilers to one of two target weights (TW): 2.1 kg (TW1) and 3.2 kg (TW2). We categorized strains by their growth rate to TW2 as conventional (CONV), fastest slow strains (FAST), moderate slow strains (MOD) and slowest slow strains (SLOW). When incubated, hatched, housed, managed and fed the same, the categories of strains differed in body weights, growth rates, feed intake and feed efficiency. At 48 days of age, strains in the CONV category were 835-1264 g heavier than strains in the other categories. By TW2, differences in body weights and feed intake resulted in a 22 to 43-point difference in feed conversion ratios. Categories of strains did not differ in their overall mortality rates.

Systematic characterization and genetic interaction analysis of adhesins in Candida albicans virulence

Rosiana, S. — 2020-10-22

Candida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, or in every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen. SummaryCandida albicans is a human fungal pathogen and cause of life-threatening systemic infections. Cell surface-associated adhesins play a central role in this pathogens ability to establish infection. Here, we provide a comprehensive analysis of adhesin factors, and their role in fungal virulence. Exploiting a high-throughput workflow, we screened an adhesin mutant library using C. elegans as a simple model host, and identified mutants and genetic interactions involved in virulence. We found that adhesin mutants are impaired in in vitro pathogenicity, irrespective of their virulence. Together, this work provides new insight into the role of adhesin factors in mediating fungal virulence.

Invasion by Fusobacterium nucleatum Causes Upregulation of dll4 and klf4 Expression in Caco-2 Cells

Allen-Vercoe, E. — 2020-10-26

Fusobacterium nucleatum is an emerging microbe of importance in the pathogenesis of colorectal cancer. Strains of this enigmatic bacterial species vary in their capacity to invade human epithelial cells, a virulence determinant which has important implications in disease. Here, we infected human colorectal epithelial (Caco-2) cells in vitro with a known, highly invasive strain of F. nucleatum isolated from a Crohns Disease patient, as well as a further invasive isolate of F. nucleatum derived from a colorectal cancer tumour. We used transcriptional profiling to determine the human genes upregulated during the invasion process compared to exposure to a non-invasive E.coli control strain. Infection with F. nucleatum strains resulted in the upregulation of several host genes, including two associated with tumorigenesis: dll4 and klf4.

Flower Power: Floral reversion as a viable alternative to nodal micropropagation in Cannabis sativa.

Monthony, A. S. — 2020-10-30

The legalization of Cannabis sativa L. for recreational and medical purposes has been gaining global momentum, leading to a rise in interest in Cannabis tissue culture as growers look for large-scale solutions to germplasm storage and clean plant propagation. Mother plants used in commercial propagation are susceptible to insect pests and disease and require considerable space. While micropropagation can produce disease free starting material in less space, current published in vitro micropropagation methods are not robust and few report high multiplication rates. Further, these micropropagation methods rely on photoperiod-sensitive plants which can be maintained in a perpetual vegetative state. Current methods are not adaptable to long-term tissue culture of day-neutral cultivars, which cannot be maintained in perpetual vegetative growth. In this study, we chose to develop a micropropagation system which uses C. sativa inflorescences as starting materials. This study used two cannabis cultivars, two plant growth regulators (PGR; 6-benzylaminopurine and meta-topolin) at different concentrations, and two different numbers of florets. Here we show that floral reversion occurs from meristematic tissue in C. sativa florets and that it can be used to enhance multiplication rates compared to existing in vitro methods. Floret number was shown to have a significant impact on percent reversion, with pairs of florets reverting more frequently and producing healthier explants than single florets, while cultivar and PGR had no significant effect on percent reversion. Compared with our previously published nodal culture studies, the current floral reversion method produced up to eight times more explants per tissue culture cycle. Floral reversion provides a foundation for effective inflorescence-based micropropagation systems in C. sativa.

Environmental DNA for tracking waterhole visitation in savanna ecosystems

Farrell, M. J. — 2020-11-05

Game parks are the last preserve of many large mammals, and in savanna ecosystems, management of surface waters poses a conservation challenge. In arid and semi-arid regions, water can be a scarce resource during dry seasons and drought. Artificial waterholes are common in parks and reserves across Africa, but can alter mammal community composition by favoring drought intolerant species, with consequences for disease dynamics, and population viability of drought-tolerant species. Analysis of waterborne environmental DNA (eDNA) is increasingly used to inform conservation of rare and invasive species, and conduct large-scale biodiversity assessments. To explore the reliability of eDNA as an indicator of mammal waterhole use in savannas, we compare eDNA metabarcoding and camera traps for documenting artificial waterhole use in the Kruger National Park, South Africa, a global hotspot for mammal diversity. We show that eDNA metabarcoding can recover the majority of mammal species detected by camera traps, including a number of endangered species, but DNA signatures of mammal visitation are temporally limited, with best performance when tracking water-dependent large bodied mammals visiting within two days of sampling. Our results highlight limitation of eDNA based monitoring in these systems, including the lack of long-term eDNA persistence in small and highly utilized waterholes, and variability in detection rates among species. However, we demonstrate that eDNA-based approaches can be used to track mammals of conservation concern, and reflect patterns of recent waterhole use and co-occurrence across water-dependent species, both of which are crucial for making evidence-based decisions regarding water management and provisioning.

Subsidy Accessibility Drives Asymmetric Food Web Responses

Gutgesell, M. K. — 2020-11-10

Global change is fundamentally altering flows of natural and anthropogenic subsidies across space and time. After a pointed call for research on subsidies in the 1990s, an industry of empirical work has documented the ubiquitous role subsidies play in ecosystem structure, stability and function. Here, we argue that physical constraints (e.g., water temperature) and species traits can govern a species accessibility to resource subsidies, and that these physical constraints have been largely overlooked in the subsidy literature. We examined the input of a high quality, point-source anthropogenic subsidy into a recipient freshwater lake food web (i.e., released net-pen aquaculture feed in Parry Sound, Lake Huron), to demonstrate the importance of subsidy accessibility in governing recipient whole food web responses. By using a combined bio-tracer approach, we detect a gradient in accessibility of the anthropogenic subsidy within the surrounding food web driven by the thermal tolerances of three constituent species. This thermally-driven accessibility gradient drives asymmetrical changes in food web structure, effectively rewiring the recipient lake food web and altering patterns in secondary production with yet unknown stability consequences. Since aquaculture is predicted to increase significantly in coming decades to support growing human populations, and global change is altering temperature regimes, then this form of food web alteration may be expected to occur frequently. We argue that subsidy accessibility is a key characteristic of recipient food web interactions that must be considered when trying to understand the impacts of subsidies on ecosystem stability and function under continued global change.

The Palmitoyl Acyltransferase ZDHHC14 Controls Kv1-Family Potassium Channel Clustering at the Axon Initial Segment

Sanders, S. S. — 2020-11-11

The palmitoyl acyltransferase (PAT) ZDHHC14 is highly expressed in the hippocampus and is the only PAT predicted to bind Type I PDZ domain-containing proteins. However, ZDHHC14s neuronal roles are unknown. Here, we identify the PDZ domain-containing Membrane-associated Guanylate Kinase (MaGUK) PSD93 as a direct ZDHHC14 interactor and substrate. PSD93, but not other MaGUKs, localizes to the Axon Initial Segment (AIS). Using lentiviral-mediated shRNA knockdown in rat hippocampal neurons, we find that ZDHHC14 controls palmitoylation and AIS clustering of PSD93 and also of Kv1 potassium channels, which directly bind PSD93. Neurodevelopmental expression of ZDHHC14 mirrors that of PSD93 and Kv1 channels and, consistent with ZDHHC14s importance for Kv1 channel clustering, loss of ZDHHC14 decreases outward currents and increases action potential firing in hippocampal neurons. To our knowledge, these findings identify the first neuronal roles and substrates for ZDHHC14 and reveal a previously unappreciated role for palmitoylation in control of neuronal excitability. Impact StatementZDHHC14 controls palmitoylation and axon initial segment targeting of PSD93 and Kv1-family potassium channels, events that are essential for normal neuronal excitability.

Degradation of boscalid, pyraclostrobin, fenbuconazole, and glyphosate residues by an advanced oxidative process utilizing ultraviolet light and hydrogen peroxide

Skanes, B. — 2020-11-15

Recently an advanced oxidative process (AOP) combining H2O2 and UV-C light was observed to be effective at controlling Listeria monocytogens (Murray et al., 2018) and Escherichia coli O157:H7 and degrading chlorpyrifos residues on the surface of apples (Ho et al., 2020). Little is known about the application of AOP for the degradation of other pesticide residues. This study examined degradation of boscalid, pyraclostrobin, fenbuconazole and glyphosate by 3% (w/v) H2O2, UV-C (254 nm) irradiation and their combination on apple skin and glass. The extent of degradation was not significantly different between the AOP and optimal individual treatment. However, treatment susceptibility was different with glyphosate most effectively degraded by H2O2 exposure (up to 98% on apple, 3% (w/v) H2O2 at 30{square}C for 15 min) while boscalid, pyraclostrobin and fenbuconazole were more effectively degraded by UV-C (up to 88%, 100% and 70% degradation after ~11,000 mJ/cm2). Suggestions for possible causes of degradation are proposed.

piNET: An Automated Proliferation Index Calculator Framework for Ki67 Breast Cancer Images

Geread, R. S. — 2020-11-20

In this work, a novel proliferation index (PI) calculator for Ki67 images called piNET is proposed. It is successfully tested on four datasets, from three scanners comprised of patches, tissue microarrays (TMAs) and wholeslide images (WSI), representing a diverse multicentre dataset for evaluating Ki67 quantification. Compared to state-of-the-art methods, piNET consistently performs the best over all datasets with an average PI difference of 5.603%, PI accuracy rate of 86% and correlation coefficient R = 0.927. The success of the system can be attributed to a number of innovations. Firstly, this tool is built based on deep learning, which can adapt to wide variability of medical images - and it was posed as a detection problem to mimic pathologists workflow which improves accuracy and efficiency. Secondly, the system is trained purely on tumour cells, which reduces false positives from non-tumour cells without needing the usual pre-requisite tumour segmentation step for Ki67 quantification. Thirdly, the concept of learning background regions through weak supervision is introduced, by providing the system with ideal and non-ideal (artifact) patches that further reduces false positives. Lastly, a novel hotspot analysis is proposed to allow automated methods to score patches from WSI that contain "significant" activity.

Debates about vaccines and climate change on social media networks: a study in contrasts

Schonfeld, J. — 2020-11-24

Vaccines and climate change have much in common. In both cases, a scientific consensus contrasts with a divided public opinion. They also exemplify coupled human-environment systems involving common pool resources. Here we used machine learning algorithms to analyze the sentiment of 87 million tweets on climate change and vaccines in order to characterize Twitter user sentiment and the structure of user and community networks. We found that the vaccine conversation was characterized by much less interaction between individuals with differing sentiment toward vaccines. Community-level interactions followed this pattern, showing less interaction between communities of opposite sentiment toward vaccines. Additionally, vaccine community networks were more fragmented and exhibited numerous isolated communities of neutral sentiment. Finally, pro-vaccine individuals overwhelmingly believed in anthropogenic climate change, but the converse was not true. We propose mechanisms that might explain these results, pertaining to how the spatial scale of an environment system can structure human populations.

Phytochrome contributes to blue-light-mediated stem elongation and associated shade-avoidance response in mature Arabidopsis plants

Zheng, Y. — 2020-11-27

Our recent studies on ornamental plants and microgreens indicate that blue-light-mediated stem elongation is related to phytochrome activity, which was based on the calculated phytochrome photoequilibrium. To examine whether phytochromes really contribute to the blue lights effect, plant phenotypic responses were investigated in wild type Arabidopsis (Col-0), and its quintuple phytochrome (phyA phyB phyC phyD phyE) mutant plants under the following light treatments: (1) R, a pure red light from 660-nm LED; (2) B, a pure blue light from 455-nm LED; (3) BR, a impure blue light from LED combination of 94% B and 6% R; and (4) BRF, another impure blue light from LED combination of BR and 6 {micro}mol m-2 s-1 of FR (735 nm). For all the light treatments, a photosynthetic photon flux density of {approx}100 mol m-2 s-1 were provided by 24-h lighting daily inside a walk-in growth chamber, which had an air temperature of {approx} 23 {degrees}C. The calculated phytochrome photoequilibrium was 0.89, 0.50, 0.69, and 0.60 for R, B, BR, and BRF, respectively, indicating a higher phytochrome activity under R and BR than B and BRF. After 18 days of light treatment, B or BRF increased main stem length in wild-type plants compared with R, but BR had an inhibition effect similar to R. Also, B and BRF relative to R or BR induced earlier flowering and reduced leaf size in wild type plants, showing typical shade-avoidance responses. In phytochrome-deficient mutant plants, the above shade-avoidance responses were inhibited under B or BRF, and induced under BR. However, as an exception, hypocotyl length, a growth trait during the de-etiolation stage, was reduced under B, BR and BRF vs. R regardless of phytochrome absence. It suggests that for mature Arabidopsis plants, phytochrome plays an active role in blue-light-mediated stem elongation and associated shade-avoidance response.

Safeguarding pollinators requires specific habitat prescriptions and substantially more land area than current policy suggests

Pindar, A. — 2020-12-15

Habitat loss and fragmentation are major drivers of global pollinator declines, yet even after recent unprecedented periods of anthropogenic land-use intensification the amount of habitat needed to support pollinators remains unknown. Here we use comprehensive datasets to determine the extent and amount of habitat needed. Safeguarding wild bee communities in a Canadian landscape requires 11.6-16.7% land-cover from a diverse range of habitats (~1.8-3.6x current policy guidelines), irrespective of whether conservation aims are enhancing species richness or abundance. Sensitive habitats, like tallgrass woodlands and wetlands, were important predictors of bee biodiversity. Conservation strategies that under-estimate the extent of habitat, spatial scale and specific habitat needs of functional guilds are unlikely to protect bee communities and the essential pollination services they provide to crops and wild plants. One sentence summarySafeguarding wild bee communities requires 11.6-16.7% of the area in common North American landscapes to provide targeted habitat prescriptions for different functional guilds over a variety of spatial scales.

Age-related reductions in the number of serial sarcomeres contribute to shorter fascicle lengths but not elevated passive tension

Power, G. A. — 2020-12-22

We investigated age-related changes to fascicle length (FL), sarcomere length (SL), and serial sarcomere number (SSN), and how this affects passive force. Following mechanical testing to determine passive force, the medial gastrocnemius muscle of young (n=9) and old (n=8) Fisher 344BN hybrid rats was chemically fixed at the optimal muscle length for force production; individual fascicles were dissected for length measurement, and laser diffraction was used to assess SL. Old rats had [~]14% shorter FL than young, which was driven by a [~]10% reduction in SSN, with no difference in SL ([~]4%). Passive force was greater in the old compared to young rats at long muscle lengths. Shorter FL and reduced SSN in the old rats could not entirely explain increased passive forces for absolute length changes, owing to a slight reduction in SL in old, resulting in similar SL at long muscle lengths. Summary StatementThis study sought to explain the increased passive tension observed for muscles of older individuals owing to age-related changes to muscle architecture.

debar, a sequence-by-sequence denoiser for COI-5P DNA barcode data

Nugent, C. M. — 2021-01-05

DNA barcoding and metabarcoding are now widely used to advance species discovery and biodiversity assessments. High-throughput sequencing (HTS) has expanded the volume and scope of these analyses, but elevated error rates introduce noise into sequence records that can inflate estimates of biodiversity. Denoising --the separation of biological signal from instrument (technical) noise--of barcode and metabarcode data currently employs abundance-based methods which do not capitalize on the highly conserved structure of the cytochrome c oxidase subunit I (COI) region employed as the animal barcode. This manuscript introduces debar, an R package that utilizes a profile hidden Markov model to denoise indel errors in COI sequences introduced by instrument error. In silico studies demonstrated that debar recognized 95% of artificially introduced indels in COI sequences. When applied to real-world data, debar reduced indel errors in circular consensus sequences obtained with the Sequel platform by 75%, and those generated on the Ion Torrent S5 by 94%. The false correction rate was less than 0.1%, indicating that debar is receptive to the majority of true COI variation in the animal kingdom. In conclusion, the debar package improves DNA barcode and metabarcode workflows by aiding the generation of more accurate sequences aiding the characterization of species diversity.

Ecological outcomes of hybridization vary extensively in Catostomus fishes

Mandeville, E. G. — 2021-01-21

Hybridization has been studied extensively to learn about speciation and mechanisms of reproductive isolation, but increasingly we recognize that hybridization outcomes vary geo-graphically and can depend on the environment. At the same time, hybridization can reshape biotic interactions in an ecosystem, leading to ecological shifts where hybridization occurs. Identifying how hybrid individuals function ecologically would link evolutionary outcomes of hybridization to ecological consequences, but relatively few studies have focused on ecological traits of hybrids. We described the feeding ecology of several Catostomus fish species and their hybrids by using stable isotopes ({delta}13C and{delta} 15N) as a proxy for diet and habitat use, and compared two native species, an introduced species, and three interspecific hybrid crosses. We replicated this comparison spatially, including hybrids and parental species from seven rivers in the Upper Colorado River basin where parental species co-occur and the opportunity for hybridization exists. Although relationships between native species in isotopic space varied, individual native species did not fully overlap in isotopic space in any river sampled, suggesting little overlap of resource use between these historically sympatric species. The introduced species overlapped with one or both native species in every river, suggesting similar resource use and potential for competition. Hybrids occupied intermediate, matching, or more extreme (transgressive) isotopic space relative to parental species, and were isotopically variable within and among rivers. We suggest that ecological outcomes of hybridization, like genomic outcomes of hybridization, are likely to vary across locations where a pair of species hybridizes. This variation implies that hybridization might have large unpredictable, idiosyncratic ecological effects on fish assemblages where hybrids occur. Although we found little evidence that hybrids are at a disadvantage ecologically--there were no significant declines in body condition relative to parental species--it is nevertheless possible that abiotic or biotic attributes of a river might constrain the range of interspecific hybrids that are successful, thus contributing to variation in hybridization outcomes across rivers.

Profile hidden Markov model sequence analysis can help remove putative pseudogenes from DNA barcoding and metabarcoding datasets

Porter, T. M. — 2021-01-26

BackgroundPseudogenes are non-functional copies of protein coding genes that typically follow a different molecular evolutionary path as compared to functional genes. The inclusion of pseudogene sequences in DNA barcoding and metabarcoding analysis can lead to misleading results. None of the most widely used bioinformatic pipelines used to process marker gene (metabarcode) high throughput sequencing data specifically accounts for the presence of pseudogenes in protein-coding marker genes. The purpose of this study is to develop a method to screen for obvious pseudogenes in large COI metabarcode datasets. We do this by: 1) describing gene and pseudogene characteristics from a simulated DNA barcode dataset, 2) show the impact of two different pseudogene removal methods on mock metabarcode datasets with simulated pseudogenes, and 3) incorporate a pseudogene filtering step in a bioinformatic pipeline that can be used to process Illumina paired-end COI metabarcode sequences. Open reading frame length and sequence bit scores from hidden Markov model (HMM) profile were used to detect pseudogenes. ResultsOur simulations showed that it was more difficult to identify pseudogenes from shorter amplicon sequences such as those typically used in metabarcoding ([~]300 bp) compared with full length DNA barcodes that are used in construction of barcode libraries ([~] 650 bp). It was also more difficult to identify pseudogenes in datasets where there is a high percentage of pseudogene sequences. We show that existing bioinformatic pipelines used to process metabarcode sequences already remove some apparent pseudogenes, especially in the rare sequence removal step, but the addition of a pseudogene filtering step can remove more. ConclusionsThe combination of open reading frame length and hidden Markov model profile analysis can be used to effectively screen out obvious pseudogenes from large datasets. There is more to learn from COI pseudogenes such as their frequency in DNA barcode and metabarcoding studies, their taxonomic distribution, and evolution. Thus, we encourage the submission of verified COI pseudogenes to public databases to facilitate future studies.

Cold-acclimation induces life stage-specific responses in the cardiac proteome of Western painted turtles (Chrysemys picta bellii): implications for anoxia tolerance

Alderman, S. L. — 2021-02-04

Western painted turtles (Chrysemys picta bellii) are the most anoxia-tolerant tetrapod. Survival time improves at low temperature and during ontogeny, such that adults acclimated to 3{degrees}C survive far longer without oxygen than either warm-acclimated adults or cold-acclimated hatchlings. Since protein synthesis is rapidly suppressed to save energy at the onset of anoxia exposure, this study tested the hypothesis that cold-acclimation would evoke preparatory changes in protein expression that would support enhanced anoxia survival in adult but not hatchling turtles. To test this, adult and hatchling turtles were acclimated to either 20{degrees}C (warm) or 3{degrees}C (cold) for 5 weeks, and then the heart ventricles were collected for quantitative proteomic analysis using labeled isobaric tags and mass spectrometry. The relative abundances of 1316 identified proteins were compared between temperatures and developmental stages. The effect of cold-acclimation on the cardiac proteome was most evident when life stage was included as a covariable, suggesting that ontogenic differences in anoxia tolerance may be predicated on successful maturation of the heart from its hatchling to adult form and, only after this maturation occurs, will cold-acclimation induce protein expression changes appropriate for supporting heart function during prolonged anoxia. The main differences between the hatchling and adult cardiac proteomes reflect an increase in metabolic scope that included more myoglobin and increased investment in both aerobic and anaerobic energy pathways. Mitochondrial structure and function were key targets of life stage- and temperature-induced changes to the cardiac proteome, including reduced complex II proteins in cold-acclimated adults that may help down-regulate the electron transport system and avoid succinate accumulation during anoxia. Therefore, targeted cold-induced changes to the cardiac proteome may be a contributing mechanism for stagespecific anoxia tolerance in turtles.

Accumulation of somatic mutations leads to genetic mosaicism in Cannabis

Adamek, K. — 2021-02-12

Cannabis is typically propagated using stem cuttings taken from mother plants to produce genetically uniform propagules. However, producers anecdotally report that clonal lines deteriorate over time and eventually produce clones with less vigour and lower cannabinoid levels than the original mother plant. While the cause of this deterioration has not been investigated, one potential contributor is the accumulation of somatic mutations within the plant. To test this, we used deep sequencing of whole genomes (>50x) to compare the variability within an individual Cannabis sativa cv. "Honey Banana" plant sampled at the bottom, middle and top. We called over 6 million sequence variants based on a reference genome and found that the top had the most by a sizable amount. Comparing the variants among the samples uncovered that nearly 600K (34%) were unique to the top while the bottom only contained 148K (12%) and middle with 77K (9%) unique variants. Bioinformatics tools were used to identify mutations in critical cannabinoid/terpene biosynthesis pathways. While none were identified as high impact, four genes contained more than double the average level of nucleotide diversity ({pi}) in or near the gene. Two genes code for essential enzymes required for the cannabinoid pathway while the other two are in the terpene pathways, demonstrating that mutations were accumulating within these pathways and could influence their function. Overall, a measurable number of intra-plant genetic diversity was discovered that could impact long-term genetic fidelity of clonal lines and potentially contribute to the observed decline in vigour and cannabinoid content.

Cancer blues? A validated judgment bias task suggests pessimism in nude mice with tumors

Resasco, A. — 2021-02-14

In humans, affective states can bias responses to ambiguous information: a phenomenon termed judgment bias (JB). Judgment biases have great potential for assessing affective states in animals, in both animal welfare and biomedical research. New animal JB tasks require construct validation, but for laboratory mice (Mus musculus), the most common research vertebrate, a valid JB task has proved elusive. Here (Experiment 1), we demonstrate construct validity for a novel mouse JB test: an olfactory Go/Go task in which subjects dig for high- or low-value food rewards. In C57BL/6 and Balb/c mice faced with ambiguous cues, latencies to dig were sensitive to high/low welfare housing, environmentally-enriched animals responding with relative optimism through shorter latencies. Illustrating the versatility of a validated JB task across fields of research, it further allowed us to test hypotheses about the mood-altering effects of cancer (Experiment 2). Male nude mice bearing subcutaneous lung adenocarcinomas responded more pessimistically than healthy controls to ambiguous cues. Similar effects were not seen in females, however. To our knowledge, this is the first validation of a mouse JB task and the first demonstration of pessimism in tumor-bearing animals. This task, especially if refined to improve its sensitivity, thus has great potential for investigating mouse welfare, the links between affective state and disease, depression-like states in animals, and hypotheses regarding the neurobiological mechanisms that underlie affect-mediated biases in judgment.

Shades of grey: Coat-colour dependent effect of urbanization on the bacterial microbiome of a wild mammal

Stothart, M. R. — 2021-02-17

BackgroundHost-associated microbiota can be fundamental to the ecology of their host and may even help wildlife species colonize novel niches or cope with rapid environmental change. Urbanization is a globally replicated form of severe environmental change which we can leverage to better understand wildlife microbiomes. Does the colonization of separate cities result in parallel changes in the intestinal microbiome of wildlife, and if so, does within-city habitat heterogeneity matter? Using 16S rRNA gene amplicon sequencing, we quantified the effect of urbanization on the microbiome of eastern grey squirrels (Sciurus carolinensis). Eastern grey squirrels are ubiquitous in both rural and urban environments throughout their native range, across which they display an apparent coat colour polymorphism (agouti, black, intermediate). ResultsGrey squirrel microbiomes differed between rural and city environments; however, comparable variation was explained by habitat heterogeneity within cities. Our analyses suggest that operational taxonomic unit (OTU) community structure was more strongly influenced by local environmental conditions (rural and city forests versus human built habitats) than urbanization of the broader landscape (city versus rural). Many of the bacterial genera identified as characterizing the microbiomes of built-environment squirrels are though to specialize on host-derived products and have been linked in previous research to low fibre diets. However, despite an effect of urbanization at fine spatial scales, phylogenetic patterns in the microbiome were coat colour phenotype dependent. City and built environment agouti squirrels displayed greater phylogenetic beta-dispersion than those in rural or forest environments, and null modelling results indicated that the phylogenetic structure of urban agouti squirrels did not differ greatly from stochastic phylogenetic expectations. ConclusionsHabitat heterogeneity at fine spatial scales affects host-associated microbiomes, however, we found little evidence that this pattern was the result of similar selective pressures acting on the microbiome within environments. Further, this result, those of phylogeny-independent analyses, and patterns of beta-dispersion lead us to suggest that microbiota dispersal and ecological drift are integral to shaping the inter-environmental differences we observed. These patterns were partly mediated by squirrel coat colour phenotype, and therefore putatively, host physiology. Given a well-known urban cline in squirrel coat colour melanism, grey squirrels provide an ideal free-living system with which to study how host genetics mediate environment x microbiome interactions.

F-type Pyocins are Diverse Non-Contractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa

Saha, S. — 2021-02-17

Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or non-contractile phage tails known as R-type and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3-end of the F-type pyocin cluster are divergent in sequence, and likely determine bactericidal specificity. We use sequence similarities among these proteins to define 11 distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly re-assorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins emerged earlier or have been subject to distinct evolutionary pressures. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as anti-bacterial therapeutics. IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes a broad spectrum of antibiotic resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great interest in the development of alternative therapeutics. One alternative is protein-based antimicrobials called bacteriocins, which are produced by one strain of bacteria to kill other strains. In this study, we investigate F-type pyocins, bacteriocins naturally produced by P. aeruginosa that resemble non-contractile phage tails. We show that they are potent killers of P. aeruginosa, and distinct pyocin groups display different killing specificities. We have identified the probable specificity determinants of F-type pyocins, which opens up the potential to engineer them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well characterized phage tails will greatly facilitate their development into effective antibacterials.

Feature-based attention induces surround suppression during the perception of visual motion

Yoo, S.-A. — 2021-02-18

Feature-based attention prioritizes the processing of the attended feature while strongly suppressing the processing of nearby ones. This creates a non-linearity or attentional suppressive surround predicted by the Selective Tuning model of visual attention. However, previously reported effects of feature-based attention on neuronal responses are linear, e.g., feature-similarity gain. Here, we investigated this apparent contradiction by neurophysiological and psychophysical approaches. Responses of motion direction-selective neurons in area MT/MST of monkeys were recorded during a motion task. When attention was allocated to a stimulus moving in the neurons preferred direction response tuning curves showed its minimum for directions 60-90 degrees away from the preferred direction, an attentional suppressive surround. This effect was modeled via the interaction of two Gaussian fields representing excitatory narrowly-tuned and inhibitory widely-tuned inputs into a neuron, with feature-based attention predominantly increasing the gain of inhibitory inputs. We further showed using a motion repulsion paradigm in humans that feature-based attention produces a similar non-linearity on motion discrimination performance. Our results link the gain modulation of neuronal inputs and tuning curves examined through the feature-similarity gain lens to the attentional impact on neural population responses predicted by the Selective Tuning model, providing a unified framework for the documented effects of feature-based attention on neuronal responses and behavior.

Solving the Sample Size Problem for Resource Selection Analysis

Street, G. M. — 2021-02-23

O_LISample size sufficiency is a critical consideration for conducting Resource-Selection Analyses (RSAs) from GPS-based animal telemetry. Cited thresholds for sufficiency include a number of captured animals M [≥] 30 and as many relocations per animal N as possible. These thresholds render many RSA-based studies misleading if large sample sizes were truly insufficient, or unpublishable if small sample sizes were sufficient but failed to meet reviewer expectations. C_LIO_LIWe provide the first comprehensive solution for RSA sample size by deriving closed-form mathematical expressions for the number of animals M and the number of relocations per animal N required for model outputs to a given degree of precision. The sample sizes needed depend on just 2 biologically meaningful quantities: habitat selection strength and a novel measure of landscape complexity, which we define rigorously. The mathematical expressions are calculable for any environmental dataset at any spatial scale and are applicable to any study involving resource selection (including sessile organisms). We validate our analytical solutions using globally relevant empirical data including 5,678,623 GPS locations from 511 animals from 10 species (omnivores, carnivores, and herbivores living in boreal, temperate, and tropical forests, montane woodlands, swamps, and arctic tundra). C_LIO_LIOur analytic expressions show that the required M and N must decline with increasing selection strength and increasing landscape complexity, and this decline is insensitive to the definition of availability used in the analysis. Our results contradict conventional wisdom by demonstrating that the most biologically relevant effects on the utilization distribution (i.e. those landscape conditions with the greatest absolute magnitude of resource selection) can often be estimated with far fewer data than is commonly assumed. C_LIO_LIWe identify several critical steps in implementing these equations, including (i) a priori selection of expected model coefficients, and (ii) sampling intensity for background (absence/pseudo-absence) data within a given definition of availability. We show that random sampling of background data violates the underlying mathematics of RSA, leading to incorrect values for necessary M and N and potentially incorrect RSA model outputs. We argue that these equations should be a mandatory component for all future RSA studies. C_LI

Plasma concentrations of progesterone in the preceding estrous cycle are associated with the intensity of estrus and fertility of Holstein cows

Madureira, A. M. L. — 2021-03-01

The aim of this study was to determine the association between concentrations of progesterone (P4) during previous the estrous cycle with the intensity of spontaneous or estrogen-induced estrous expression and pregnancy per artificial insemination (P/AI). A total of 1,953 AI events from lactating Holstein cows were used, consisting of 1,289 timed AI events from experiment 1 (Exp. 1) and 664 AI events from experiment 2 (Exp. 2). In Exp. 1, cows were bred after a timed AI protocol based on estradiol and P4. In Exp. 2 animals were bred upon spontaneous estrus detection. In both experiments cows were continuously monitored by an automated activity monitor (AAM). The intensity of estrous expression was determined for each event and classified as either high or low intensity using the median of each experiment. Blood samples were collected for P4 analysis in Exp. 1 at -4 d, 0 d, and 7 d relative to timed AI, and in Exp. 2 immediately following estrus (0 d), 7 d, 14 d, and 21 d post-AI. Concentration of P4 was classified as greater or lower according to the median value in each experiment. Cows with lower concentrations of P4 at AI had greater estrous expression in Exp. 1 (363.6 {+/-} 5.2 vs. 275.9 {+/-} 8.0 % relative increase) and Exp. 2 (76.7 {+/-} 1.9 vs. 67.4 {+/-} 4.7 index; and 12.5 {+/-} 0.5 vs. 9.3 {+/-} 1.8 h). Cows with a greater intensity of estrous expression at timed AI had greater concentrations of P4 at -4 d than cows with lower intensity estrus or no estrous expression (4.6 {+/-} 0.2 vs. 3.6 {+/-} 0.2 vs. 3.7 {+/-} 0.2 ng/mL). Cows with greater concentrations of P4 at -4 d had greater P/AI (32.8 {+/-} 4.4 vs. 22.4 {+/-} 4.5 %), whereas cows with lesser concentrations of P4 at d0 for either timed AI (35.2 {+/-} 3.4 vs. 19.6 {+/-} 4.6 %) or spontaneous estrus (31.8 {+/-} 2.8 vs. 23.4 {+/-} 3.2 %) had greater P/AI. Cows with greater concentrations of P4 7 d post-timed AI had greater P/AI compared with cows that had lower concentration of P4 (39.1 {+/-} 2.9 vs. 24.7 {+/-} 2.6 %). Similarly, cows that had lower concentrations of P4 at 7 d, 14 d and 21 d post-spontaneous estrus tended to have lower P/AI when compared with cows with greater concentrations of P4. Overall, concentrations of P4 prior to and at AI were associated with greater estrous intensity and P/AI at both spontaneous and timed AI events.

Revision and annotation of DNA barcode records for marine invertebrates: report of the 8th iBOL conference hackathon

Radulovici, A. E. — 2021-03-08

The accuracy of the identification of unknown specimens using DNA barcoding and metabarcoding relies on reference libraries containing records with reliable taxonomy and sequence quality. A rampant growth in barcode data led to a stringent need for data curation, especially in taxonomically difficult groups such as marine invertebrates. A major effort in curating marine barcode data deposited in the Barcode of Life Data Systems (BOLD) has been undertaken during the 8th International Barcode of Life Conference (Trondheim, Norway, 2019). For practical reasons, only major taxonomic groups were reviewed and annotated (crustaceans, echinoderms, molluscs, and polychaetes). The congruence of Linnean names with Barcode Index Numbers (BINs) was investigated, and the records deemed uncertain were annotated with four tags: a) MIS-ID (misidentified, mislabeled or contaminated records), b) AMBIG (ambiguous records unresolved with the current data), c) COMPLEX (species occurring in multiple BINs), and d) SHARE (barcodes shared between species). A total of 83,712 specimen records corresponding to 7,576 species were reviewed and 39% of the species were tagged (7% MIS-ID, 17% AMBIG, 14% COMPLEX, and 1% SHARE). High percentages (>50%) of AMBIG tags were recorded in gastropods, whereas COMPLEX tags dominated in crustaceans and polychaetes. This high proportion of tagged species reflects either flaws in the barcoding workflow (e.g., misidentification, cross -contamination) or taxonomic difficulties (e.g., synonyms, undescribed species). Although data curation is crucial for barcode applications, such manual efforts of reviewing large datasets are not sustainable and the implementation of automated solutions to the furthest possible extent is hi ghly desirable.

The SAGA and NuA4 component Tra1 regulates Candida albicans drug resistance and pathogenesis

Razzaq, I. — 2021-03-18

Candida albicans is the most common cause of death from fungal infections. Emergence of resistant strains reducing the efficacy of first line therapy with echinocandins such as caspofungin calls for the identification of alternative therapeutic strategies. Tra1 is an essential component of the SAGA and NuA4 transcriptional co-activator complexes. As a PIKK family member, Tra1 is characterized by a C-terminal phosphoinositide 3-kinase domain. In Saccharomyces cerevisiae, the assembly and function of SAGA and NuA4 is compromised by a version of Tra1 (Tra1Q3) with three arginine residues in the putative ATP-binding cleft changed to glutamine, Whole transcriptome analysis of the S. cerevisiae tra1Q3 strain highlights Tra1s role in global transcription, stress response and cell wall integrity. As a result, tra1Q3 increases susceptibility to multiple stressors, including caspofungin. Moreover, the same tra1Q3 allele in the pathogenic yeast Candida albicans causes similar phenotypes, suggesting that Tra1 broadly mediates the antifungal response across yeast species. Transcriptional profiling in C. albicans identified 68 genes that were differentially expressed when the tra1Q3 strain was treated with caspofungin, as compared to gene expression changes induced by either tra1Q3 or caspofungin alone. Included in this set were genes involved in cell wall maintenance, adhesion and filamentous growth. Indeed, the tra1Q3 allele reduces filamentation and other pathogenesis traits in C. albicans. We identified EVP1, which encodes a putative plasma membrane protein, amongst the Tra1-regulated genes, Disrupting EVP1 results in reduced filamentation and infection capacity in C. albicans. Thus,Tra1 emerges as a promising therapeutic target for fungal infections. ImportanceFungal pathogens such as Candida albicans are important agents of infectious disease, with increasing rates of drug resistance, and limited available antifungal therapeutics. In this study, we characterize the role of C. albicans Tra1, a critical component of acetyltransferase complexes, involved in transcriptional regulation and responses to environmental stress. We find C. albicans genetic mutants with impaired Tra1 function have reduced tolerance to cell-wall targeting stressors, including the clinically-important antifungal caspofungin. We further use RNA-sequencing to profile the global fungal response to the tra1 mutation, and identify a previously uncharacterized C. albicans gene, EVP1. We find that both TRA1 and EVP1 play an important role in phenotypes associated with fungal pathogenesis, including cellular morphogenesis, biofilm formation, and toxicity towards host immune cells. Together, this work describes the key role for Tra1 in regulating fungal drug tolerance and pathogenesis, and positions this protein as a promising therapeutic target for fungal infections.

Cation and anion channelrhodopsins: Sequence motifs and taxonomic distribution

Govorunova, E. G. — 2021-03-23

Cation and anion channelrhodopsins (CCRs and ACRs, respectively) primarily from two algal species, Chlamydomonas reinhardtii and Guillardia theta, have become widely used as optogenetic tools to control cell membrane potential with light. We mined algal and other protist polynucleotide sequencing projects and metagenomic samples to identify 75 channelrhodopsin homologs from three channelrhodopsin families, including one revealed in dinoflagellates in this study. We carried out electrophysiological analysis of 33 natural channelrhodopsin variants from different phylogenetic lineages and 10 metagenomic homologs in search of sequence determinants of ion selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins. Our results show that association of a reduced number of glutamates near the conductance path with anion selectivity depends on a wider protein context, because prasinophyte homologs with the identical glutamate pattern as in cryptophyte ACRs are cation-selective. Desensitization is also broadly context-dependent, as in one branch of stramenopile ACRs and their metagenomic homologs its extent roughly correlates with phylogenetic relationship of their sequences. Regarding spectral tuning, two prasinophyte CCRs exhibit red-shifted spectra to 585 nm, although their retinal-binding pockets do not match those of previously known similarly red-shifted channelrhodopsins. In cryptophyte ACRs we identified three specific residue positions in the retinal-binding pocket that define the wavelength of their spectral maxima. Lastly, we found that dinoflagellate rhodopsins with a TCP motif in the third transmembrane helix and a metagenomic homolog exhibit channel activity. IMPORTANCEChannelrhodopsins are widely used in neuroscience and cardiology as research tools and are considered as prospective therapeutics, but their natural diversity and mechanisms remain poorly characterized. Genomic and metagenomic sequencing projects are producing an ever-increasing wealth of data, whereas biophysical characterization of the encoded proteins lags behind. In this study we used manual and automated patch clamp recording of representative members of four channelrhodopsin families including a family that we report in this study in dinoflagellates. Our results contribute to a better understanding of molecular determinants of ionic selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins.

Rescue of aberrant huntingtin palmitoylation ameliorates mutant huntingtin-induced toxicity

Lemarie, F. L. — 2021-03-28

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion in the HTT gene that codes for an elongated polyglutamine tract in the huntingtin (HTT) protein. HTT is subject to multiple post-translational modifications (PTMs) that regulate its cellular function. Mutating specific PTM sites within mutant HTT (mHTT) in HD mouse models can modulate disease phenotypes, highlighting the key role of HTT PTMs in the pathogenesis of HD. These findings have led to increased interest in developing small molecules to modulate HTT PTMs in order to decrease mHTT toxicity. However, the therapeutic efficacy of pharmacological modulation of HTT PTMs in preclinical HD models remains largely unknown. HTT is palmitoylated at cysteine 214 by the huntingtin-interacting protein 14 (HIP14 or ZDHHC17) and 14-like (HIP14L or ZDHHC13) acyltransferases. Here, we assessed if HTT palmitoylation should be regarded as a therapeutic target to treat HD by (1) investigating palmitoylation dysregulation in rodent and human HD model systems, (2) measuring the impact of mHTT-lowering therapy on brain palmitoylation, and (3) evaluating if HTT palmitoylation can be pharmacologically modulate. We show that palmitoylation of mHTT and some HIP14/HIP14L-substrates is decreased early in multiple HD mouse models, and that aging further reduces HTT palmitoylation. Lowering mHTT in the brain of YAC128 mice is not sufficient to rescue aberrant palmitoylation. However, we demonstrate that mHTT palmitoylation can be normalized in COS-7 cells, in YAC128 cortico-striatal primary neurons and HD patient-derived lymphoblasts using an acyl-protein thioesterase (APT) inhibitor. Moreover, we show that modulating palmitoylation reduces mHTT aggregation and mHTT-induced cytotoxicity in COS-7 cells and YAC128 neurons. HighlightsO_LIPalmitoylation of mHTT is reduced in multiple transgenic HD mouse models C_LIO_LIHTT palmitoylation decreases with increasing polyQ length in HD patient cells C_LIO_LImHTT-lowering in mouse brains does not rescue aberrant palmitoylation C_LIO_LImHTT palmitoylation in HD patient-derived cells can be rescued via APT inhibition C_LIO_LIPromoting palmitoylation reduces mHTT aggregation and cytotoxicity in vitro C_LI

Paternal obesity results in placental hypoxia and sex-specific impairments in placental vascularization and offspring metabolic function

Jazwiec, P. A. — 2021-03-29

Paternal obesity predisposes offspring to metabolic dysfunction, but the underlying mechanisms remain unclear. We investigated whether paternal obesity-induced offspring metabolic dysfunction is associated with placental endoplasmic reticulum (ER) stress and impaired vascular development. We determined whether offspring glucose intolerance is fueled by ER stress-mediated changes in fetal hepatic development. Furthermore, we also determined whether paternal obesity may indirectly affect in utero development by disrupting maternal metabolic adaptations to pregnancy. Male mice fed a standard chow diet (CON; 17% kcal fat) or high fat diet (PHF; 60% kcal fat) for 8-10 weeks were time-mated with control female mice to generate pregnancies and offspring. Glucose tolerance in pregnant females was evaluated at mid-gestation (embryonic day (E) 14.5) and term gestation (E18.5). At E14.5 and E18.5, fetal liver and placentae were collected, and markers of hypoxia, angiogenesis, endocrine function, and macronutrient transport, and unfolded protein response (UPR) regulators were evaluated to assess ER stress. Young adult offspring glucose tolerance and metabolic parameters were assessed at [~]60 days of age. Paternal obesity did not alter maternal glucose tolerance or placental lactogen in pregnancy but did induce placental hypoxia, ER stress, and altered placental angiogenesis. This effect was most pronounced in placentae associated with female fetuses. Consistent with this, paternal obesity also activated the ATF6 and PERK branches of the UPR in fetal liver and altered hepatic expression of gluconeogenic factors at E18.5. Adult offspring of obese fathers showed glucose intolerance and impaired whole-body energy metabolism, particularly in female offspring. Thus, paternal obesity programs sex-specific adverse placental structural and functional adaptations and alters fetal hepatic development via ER stress-induced pathways. These changes likely underpin metabolic deficits in adult offspring. Summary SentencePaternal obesity alters placental vascular structures and is associated with sex-specific compromises in glucose tolerance and metabolism in young offspring

Deep learning for early warning signals of regime shifts

Bury, T. — 2021-03-29

Many natural systems exhibit regime shifts where slowly changing environmental conditions suddenly shift the system to a new and sometimes very different state. As the tipping point is approached, the dynamics of complex and varied systems all simplify down to a small number of possible normal forms that determine how the new regime will look. Indicators such as increasing lag-1 autocorrelation and variance provide generic early warning signals (EWS) by detecting how dynamics slow down near the tipping point. But they do not indicate what type of new regime will emerge. Here we develop a deep learning algorithm that can detect EWS in systems it was not explicitly trained on, by exploiting information about normal forms and scaling behaviour of dynamics near tipping points that are common to many dynamical systems. The algorithm detects EWS in 268 empirical and model time series from ecology, thermoacoustics, climatology, and epidemiology with much greater sensitivity and specificity than generic EWS. It can also predict the normal form that will characterize the oncoming regime shift. Such approaches can help humans better manage regime shifts. The algorithm also illustrates how a universe of possible models can be mined to recognize naturally-occurring tipping points.

Perception of effort during an isometric contraction is influenced by prior muscle lengthening or shortening

Kozlowski, B. — 2021-03-30

PurposeFollowing a shortening or lengthening muscle contraction, the torque produced in the isometric steady state is distinctly lower (residual torque depression; rTD) or higher (residual torque enhancement; rTE), respectively, compared to a purely isometric contraction at the same final muscle length and level of activation. This is referred to as the history dependence of force. When matching a given torque level, there is greater muscle activation (electromyography; EMG) following shortening and less activation following lengthening. Owing to these differences in neuromuscular activation, it is unclear whether perception of effort is altered by the history dependence of force. MethodsExperiment 1 tested whether perception of effort differed between the rTD and rTE state when torque was matched. Experiment 2 tested whether perception of effort differed between the rTD and rTE state when EMG was matched. Finally, experiment 3 tested whether EMG differed between the rTD and rTE state when perception of effort was matched. ResultsWhen torque was matched, both EMG and perception of effort were higher in the rTD compared to rTE state. When EMG was matched, torque was lower in the rTD compared to rTE state while perception of effort did not differ between the two states. When perception of effort was matched, torque was lower in the rTD compared to rTE state and EMG did not differ between the two states. ConclusionThe combined results from these experiments indicate that the history dependence of force alters ones perception of effort, dependent on the level of motor command.

Phosphorylation-dependent control of Arc protein by synaptic plasticity regulator TNIK

Walczyk Mooradally, A. — 2021-04-04

Activity-regulated cytoskeleton-associated protein (Arc) is an immediate-early gene product that support neuroplastic changes important for cognitive function and memory formation. As a protein with homology to the retroviral Gag protein, a particular characteristic of Arc is its capacity to self-assemble into virus-like capsids that can package mRNAs and transfer those transcripts to other cells. Although a lot has been uncovered about the contributions of Arc to neuron biology and behavior, very little is known about how different functions of Arc are coordinately regulated both temporally and spatially in neurons. The answer to this question we hypothesized must involve the occurrence of different protein post-translational modifications acting to confer specificity. In this study, we used mass spectrometry and sequence prediction strategies to map novel Arc phosphorylation sites. Our approach led us to recognize serine 67 (S67) and threonine 278 (T278) as residues that can be modified by TNIK, which is a kinase abundantly expressed in neurons that shares many functional overlaps with Arc and has, along with its interacting proteins such as the NMDA receptor, been implicated as a risk factor for psychiatric disorders. Furthermore, characterization of each residue using site-directed mutagenesis to create S67 and T278 mutant variants revealed that TNIK action at those amino acids can strongly influence Arcs subcellular distribution and self-assembly as capsids. Together, our findings reveal an unsuspected connection between Arc and TNIK. Better understanding of the interplay between these two proteins in neuronal cells could lead to new insights about apparition and progression of psychiatric disorders.

Multiscale interactome analysis coupled with off-target drug predictions reveals drug repurposing candidates for human coronavirus disease

Sugiyama, M. — 2021-04-13

The COVID-19 pandemic has led to an urgent need for the identification of new antiviral drug therapies that can be rapidly deployed to treat patients with this disease. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of COVID-19. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cellbased experimental assessment reveals several clinically-relevant repurposing drug candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.

Diet composition of reintroduced Red-and-Green Macaws (Ara chloropterus) reflects gradual adaption to life in the wild

Volpe, N. L. — 2021-04-15

Over the last two centuries, the Red-and-green Macaw (Ara chloropterus) has become locally extinct in Argentina. In an attempt to restore its key ecosystem functions as both disperser and regulator of large-seeded plants, a reintroduction project was initiated at the Ibera National Park in northeastern Argentina. The ability of released individuals to find food is crucial, in particular when working with captive-bred animals, as long-term establishment of a self-sustaining population depends on their short-term ability to exploit wild food sources. Monitoring of feeding habits is usually conducted through behavioral observation, but in recent years DNA metabarcoding has emerged as an alternative for obtaining highly resolved data on diet composition. In this study we use a combination of both techniques to characterize the breadth and composition of the reintroduced macaws diet. In addition, we evaluate the efficiency of both direct field observations and metabarcoding of feces as techniques to assess diet composition. Individuals fed on a variety of plant species (n = 49) belonging to a broad phylogenetic spectrum (28 families). Dietary richness estimated by direct observation and metabarcoding was similar, though smaller than the combination of the two datasets as both techniques detected at least 15 species not recorded by the other method. While the total number of detected species was higher for observational data, the rate of species-detection per sampling day was higher for metabarcoding. These results suggest that a combination of both methods is required in order to obtain the most accurate account of the total diversity of the diet of a species. The ability of the reintroduced macaws to successfully exploit local food resources throughout the year indicates a good level of adjustment to the release site, an important step towards the creation of a stable, self-sustaining population of Red-and-green Macaws in Northern Argentina. RESUMENEn el transcurso de los ultimos dos siglos, el Guacamayo Rojo (Ara chloropterus) se ha extinguido en la Argentina. Buscando recuperar su rol ecologico tanto de dispersor como de depredador de semillas de gran tamano, se comenzo un proyecto de reintroduccion de la especie en el Parque Nacional Ibera, en la region noreste del pais. La capacidad para encontrar alimento por parte de los individuos liberados es crucial, particularmente cuando se trabaja con animales provenientes de condiciones de cautiverio, ya que el establecimiento de una poblacion autosuficiente a largo plazo dependera de la habilidad de estos para explotar fuentes de alimento silvestre a corto plazo. El monitoreo de habitos alimenticios se realiza usualmente a traves de observaciones comportamentales. Sin embargo, en los ultimos anos la tecnica del meta-codigo de barras de ADN ha surgido como una alternativa para la obtencion de datos de composicion dietaria con alto nivel de resolucion. En este estudio, utilizamos una combinacion de ambas tecnicas para caracterizar la amplitud y composicion de la dieta de los guacamayos reintroducidos. A su vez, evaluamos la eficiencia de la observacion directa y el codigo de barras genetico de heces como tecnicas para evaluar la composicion de la dieta. Los individuos se alimentaron de una amplia variedad de especies (n = 49), abarcando un amplio espectro filogenetico (28 familias). La riqueza dietaria estimada por observacion directa y por meta-codigo de barras genetico fue similar, aunque menor a la resultante de la combinacion de todos los datos ya que ambas tecnicas detectaron al menos 15 especies no registradas por el otro metodo. Mientras que el numero total de especies detectadas fue mayor para los metodos observacionales, la tasa de deteccion de especies por dia de muestreo fue mayor para el analisis genetico. Estos resultados sugieren que una combinacion de ambos metodos es necesaria para obtener la descripcion mas precisa posible de la diversidad dietaria total de una especie. La capacidad de los guacamayos reintroducidos para explotar recursos alimenticios locales a lo largo del ano estaria indicando un buen nivel de adaptacion al sitio de liberacion, un paso muy importante hacia el establecimiento de una poblacion de Guacamayo Rojo estable y autosuficiente en el norte de Argentina. Palabras claveAra chloropterus, Conservacion, Dieta, Frugivoria, Meta-codigo de barras, Guacamayo Rojo, Reintroduccion, Ecologia trofica LAY SUMMARYO_LIThe Red-and-green Macaw reintroduction project aims to restore this species to Argentina, where it is locally extinct. To assess if reintroduced macaws are successfully adapting to life in the wild, we studied their foraging habits at the Ibera National Park. Their food consumption was observed visually, and their feces were analyzed with molecular methods for traces of the consumed plants. C_LIO_LIMacaws fed from a large diversity of food items, exhibiting a flexible diet which varied with fruit availability in different months. A combination of both methods was required to obtain the most accurate account of the total diversity of the diet of a species. C_LIO_LIThe reintroduced macaws were able to successfully locate and exploit food resources throughout the year, indicating a good level of adjustment to the release site. C_LI

Costs, Benefits, Parasites and Mutualists: The Use and Abuse of the Mutualism-Parasitism Continuum Concept for Epichloë Fungi

Newman, J. A. — 2021-04-22

The species comprising the fungal endophyte genus Epichloe are symbionts of cool season grasses. About half the species in this genus are strictly vertically transmitted, and evolutionary theory suggests that these species must be mutualists. Nevertheless, Faeth and Sullivan (e.g. 2003) have argued that such vertically transmitted endophytes are "usually parasitic," and Muller and Krauss (2005) have argued that such vertically transmitted endophytes fall along a mutualism-parasitism continuum. These papers (and others) have caused confusion in the field. We used a systematic review to show that close to half of the published papers in this field incorrectly categorize the interaction. Here, we develop the argument that advantages and disadvantages are not the same things as mutualism and parasitism and that experimental evidence must be interpreted in the context of theory. We argue that, contra Faeth and Sullivan, it is highly unlikely that such strictly vertically transmitted endophytes can be parasites, and that, contra Muller and Krauss, it is inappropriate to apply the continuum concept to strictly vertically transmitted endophytes. We develop a mathematical model to support and illustrate our arguments. We use the model to clarify that parasitism requires some degree of horizontal transmission, and that while it is appropriate to use the continuum concept when referring to the genus as a whole, or to species that possess horizontal transmission, we argue that it is inappropriate to use the concept when referring to species that are strictly vertically transmitted.

Cannabis Vapour Exposure Alters Neural Circuit Oscillatory Activity In A Neurodevelopmental Model Of Schizophrenia: Exploring The Differential Impact Of Cannabis Constituents

Jenkins, B. — 2021-04-29

Cannabis use is highly prevalent in patients with schizophrenia and worsens the course of the disorder. To understand the causal impacts of cannabis on schizophrenia-related oscillatory disruptions, we herein investigated the impact of exposure to cannabis vapour (containing delta-9-tetrahydrocannabinol [THC] or balanced THC and cannabidiol [CBD]) on oscillatory activity in the neonatal ventral hippocampal lesion (NVHL) rat model of schizophrenia. Male Sprague Dawley rats underwent NVHL or sham surgeries on postnatal day 7. In adulthood, electrodes were implanted targeting the cingulate cortex (Cg), the prefrontal cortex (PFC), the dorsal hippocampus (HIP), and the nucleus accumbens (NAc). Local field potential recordings were obtained following exposure to two strains of vapourized cannabis flower (with ~10% THC or ~10% balanced THC:CBD) in a cross-over design with a two-week wash-out period between exposures. Compared to controls, NVHL rats had reduced baseline gamma power in the Cg, dHIP, and NAc, and reduced high-gamma coherence between the dHIP-Cg. THC-only vapour broadly suppressed oscillatory power and coherence, even beyond the baseline suppressions observed in NHVL rats. Balanced THC:CBD vapour appeared to ameliorate the THC-induced impacts on power and coherence in both sham and NVHL rats. For NVHL rats, THC-only vapour also normalized the baseline dHIP-Cg high-gamma coherence deficits. NHVL rats also demonstrated a 20ms delay in dHIP theta to high-gamma phase coupling, which was ameliorated by both exposures in the PFC and NAc. In conclusion, THC-only cannabis vapour suppressed oscillatory activity in NVHL and sham rats, while balanced THC:CBD vapour may ameliorate some of these effects.

ePlant in 2021: New Species, Viewers, Data Sets, and Widgets

Waese-Perlman, B. — 2021-04-29

ePlant was introduced in 2017 for exploring large Arabidopsis thaliana data sets from the kilometre to nanometre scales. In the past four years we have used the ePlant framework to develop ePlants for 15 agronomically-important species: maize, poplar, tomato, Camelina sativa, soybean, potato, barley, Medicago truncatula, eucalyptus, rice, willow, sunflower, Cannabis sativa, wheat and sugarcane. We also updated the interface to improve performance and accessibility, and added two new views to the Arabidopsis ePlant - the Navigator and Pathways viewers. The former shows phylogenetic relationships between homologs in other species and their expression pattern similarities, with links to view data for those genes in the respective ePlants. The latter shows Plant Reactome metabolic reactions. We also describe new Arabidopsis data sets including single cell RNA-seq data from roots, and how to embed ePlant eFP expression pictographs into any web page.

A murine model of the human CREBRFR457Q obesity-risk variant does not influence energy or glucose homeostasis in response to nutritional stress

Kershaw, E. E. — 2021-05-06

Obesity and diabetes have strong heritable components, yet the genetic contributions to these diseases remain largely unexplained. In humans, a missense variant in Creb3 regulatory factor (CREBRF) [rs373863828 (p.Arg457Gln); CREBRFR457Q] is strongly associated with increased odds of obesity but decreased odds of diabetes. Although virtually nothing is known about CREBRFs mechanism of action, emerging evidence implicates it in the adaptive transcriptional response to nutritional stress downstream of TORC1. The objectives of this study were to generate a murine model with knockin of the orthologous variant in mice (CREBRFR458Q) and to test the hypothesis that this CREBRF variant promotes obesity and protects against diabetes by regulating energy and glucose homeostasis downstream of TORC1. To test this hypothesis, we performed extensive phenotypic analysis of CREBRFR458Q knockin mice at baseline and in response to acute (fasting/refeeding), chronic (low- and high-fat diet feeding), and extreme (prolonged fasting) nutritional stress as well as with pharmacological TORC1 inhibition. The results demonstrate that the murine CREBRFR458Q model of the human CREBRFR457Q variant does not influence energy/glucose homeostasis in response to these interventions. Alternative preclinical models and/or studies in humans will be required to decipher the mechanisms linking this variant to human health and disease.

Mitogen-activated protein kinases contribute to temperature induced cardiac remodelling in rainbow trout (Oncorhynchus mykiss) heart

Ding, Y. — 2021-05-12

Rainbow trout (Oncorhynchus mykiss) live in temperate environments and experience seasonal changes in temperature that range between 4{degrees}C and 20{degrees}C. Laboratory studies demonstrate that cold and warm acclimation of male trout can have oppositional effects on cardiac hypertrophy and the collagen content of the heart. The cellular mechanisms behind temperature induced cardiac remodelling are unclear, as is why this response differs between male and female fish. Recent work utilizing cultured trout cardiac fibroblasts suggests that collagen deposition is regulated, at least in part, by mitogen-activated protein kinase (MAPK) cell signalling pathways. We therefore hypothesized that temperature-dependent cardiac remodelling is regulated by these same cell signalling pathways. To test this, male and female trout were acclimated to 18{degrees}C (warm) in the summer and to 4{degrees}C (cold) in the winter and the activation of MAPK pathways in the hearts were characterized and compared to that of control fish maintained at 12{degrees}C. Animals, maintained under a natural photoperiod matched to time of year, were sampled throughout each acclimation. p38 MAPK phosphorylation increased in the hearts of female fish during the cold acclimation protocol and the phosphorylation of extracellular signal-regulated kinase (ERK) increased in the hearts of male fish with warm acclimation. These results indicate that thermal acclimation has transient and sex-specific effects on the phosphorylation of MAPKs.

In pursuit of a better broiler: A comparison of the inactivity, behavior, and enrichment use of fast- and slower-growing broiler chickens

Dawson, L. C. — 2021-05-20

Selection for rapid growth has produced heavier, more efficient broiler chickens, but has also introduced health and welfare issues, which may cause or be caused by inactivity. Rapid growth may also limit the performance of motivated behaviors, whereas the provision of enrichment may increase these behaviors and general activity. This study aimed to evaluate the inactivity, behavior, and enrichment use of 2 fast- (CONV) and 12 slower-growing broiler strains [categorized as fastest (FAST), moderate (MOD) and slowest slow (SLOW), based on their growth rates]. To evaluate inactivity, one male and one female from 153 pens were outfitted with omni-directional accelerometers from day 21 until processing (14-24 birds/strain from 8-12 pens/strain). Additionally, to supplement inactivity data, five-minute continuous behavioral observations of four focal birds per pen (2 males, 2 females) were conducted on days 26, 42, and 56 (72-148 observations of 8-12 pens/strain) to quantify the duration and frequency of various behaviors; at the same time, 5-11 instantaneous scan samples were also performed for pen-based enrichment use. Inactivity peaked at 78-80% of the day for all strains; however, those with slower growth rates reached these levels at older ages. Compared to slower-growing strains at the same age, faster-growing strains were more inactive, spent more time sitting and feeding, spent less time standing and walking, and used enrichments less; these differences mostly occurred at younger ages. Generally, at the same age, strains with similar growth rates (within the same category) behaved similarly, with only a few exceptions. Results suggest that not all strains identified as slow-growing broilers behave differently from fast-growing broilers, nor do they all behave similarly to each other. As such, results suggest that improved broiler welfare, particularly with respect to reduced inactivity, the performance of a wider range of normal, motivated behaviors, and/or increased enrichment use, is related to the broiler strains specific growth rate.

Transcriptomics of improved fruit retention by hexanal in 'Honeycrisp' reveals hormonal crosstalk and reduced cell-wall remodelling in the fruit-abscission zone

Sriskantharajah, K. — 2021-05-20

Apples (Malus domestica Borkh) are prone to pre-harvest fruit drop which is more pronounced in Honeycrisp. Using a transcriptomic approach, we analyzed the molecular mechanisms of fruit retention in Honeycrisp. A total of 726 differentially expressed genes (DEGs) were identified in the abscission zone of hexanal-treated and untreated fruit (FAZ). Hexanal down-regulated the genes involved in ethylene biosynthesis, such as S-adenosylmethionine synthase (SAM2) and 1-aminocyclopropane-1carboxylic acid oxidases (ACO3, ACO4 and ACO4-like). Genes related to ABA biosynthesis (FDPS and CLE25) were also down-regulated. On the contrary, gibberellic acid (GA) biosynthesis genes, gibberellin 20 oxidase1-like (GA20OX-like) and ent-kaurene oxidase (KO) were up-regulated. Further, hexanal down-regulated the expression of genes related to cell-wall remodelling enzymes such as polygalacturonase (PG1), glucanases (endo-{beta}-1,4-glucanase; EG) and expansins (EXPA1-like, EXPA6, EXPA8, EXPA10-like, EXPA16-like). Hexanal also reduced ethylene, and abscisic acid (ABA) production at commercial harvest stage. Hexanal reduced ethylene production in fruits and thus reduced the sensitivity of FAZ cells to ethylene and ABA. Simultaneously, hexanal maintained the cell-wall integrity of FAZ cells by regulating genes involved in cell-wall modifications. Our findings show that fruit abscission is delayed by hexanal, by down regulating ABA through an ethylene-dependent mechanism. HighlightHexanal, a naturally occurring plant compound, increased fruit retention in apples by decreasing ethylene and ABA production and maintaining the cell-wall integrity in the fruit abscission zone.

Personality profiling may help select better cleaner fish for sea-lice control in salmon farming

Whittaker, B. A. — 2021-05-22

Lumpfish (Cyclopterus lumpus) are increasingly being used as cleaner fish to control parasitic sea-lice in salmon farming, but cleaning rates are very variable and not all individuals eat sea-lice, which increases the risk of emaciation and has ethical and practical implications. Selecting good cleaners is a priority to make the industry more sustainable, but there is little information on what behaviours make cleaner fish effective under a commercial setting. We examined variation in lumpfish personalities according to the five-factor personality model that takes into account differences in activity, anxiety (shelter use, thigmotaxis), aggression, sociality, and boldness (neophobia). We then quantified how variation in lumpfish personalities influenced interactions with naive Atlantic salmon (Salmo salar), without the confounding effects of variation in sea-lice loads. Variation in activity, sociality, aggression and neophobia, but not in anxiety, was repeatable, which is consistent with a heritable basis. Neophilic, non-aggressive lumpfish spent more time inspecting salmon than neophobic and aggressive individuals, but salmon fled in the presence of the most active and social individuals, suggesting there may be an optimal cleaner fish personality amenable to artificial selection. The personality screening protocols developed in this study could inform a more efficient use of cleaner fish in salmon farming and reduce the number of individuals required to control sea-lice.

Modulation of the host cell transcriptome and epigenome by Fusobacterium nucleatum

Despins, C. A. — 2021-05-28

Fusobacterium nucleatum (Fn) is a ubiquitous opportunistic pathogen with an emerging role as an oncomicrobe in colorectal and other cancer types. Fn can adhere to and invade host cells in a manner that varies across Fn strains and host cell phenotypes. Here we performed pairwise co-cultures between three Fn strains and two immortalized primary host cell types (colonic epithelial cells and vascular endothelial cells) followed by RNA-seq and ChIP-seq to investigate transcriptional and epigenetic host cell responses. We observed that Fn-induced host cell transcriptional modulation involves strong upregulation of genes related to immune migration and inflammatory processes, such as TNF, CXCL8, CXCL1, and CCL20. Further, we identified genes strongly upregulated specifically in conditions of host cell invasion, including overexpression of both EFNA1 and LIF, two genes commonly upregulated in colorectal cancer and associated with poor patient outcomes, and PTGS2 (COX2), a gene associated with the protective effect of aspirin in the colorectal cancer setting. Interestingly, we also observed downregulation of numerous histone modification genes upon Fn exposure. To further explore this relationship, we used the ChIP-seq data to annotate chromatin states genome-wide. We found significant chromatin remodeling following Fn exposure in conditions of host cell invasion, with substantial increases in the frequency of states corresponding to active enhancers as well as low signal or quiescent states. Thus, our results highlight increased inflammation and chemokine gene expression as conserved host cell responses to Fn exposure, and extensive host cell epigenomic changes associated with Fn host cell invasion. These results extend our understanding of Fn as an emerging pathogen and highlight the importance of considering strain heterogeneity and host cell phenotypic variation when exploring pathogenic mechanisms of Fn.

Catheterized-bladder environment induces hyphal Candida albicans formation, promoting fungal colonization and persistence.

La Bella, A. A. — 2021-06-02

Catheter-associated urinary tract infections (CAUTIs) account for 40% of all hospital-acquired infections. Given that 20-50% of all hospitalized patients receive a catheter, CAUTIs are one of the most common hospital-acquired infections and a significant medical complication as they result in increased morbidity, mortality, and an estimated annual cost of $340-370 million. Candida spp. - specifically Candida albicans - are a major causative agent of CAUTIs (17.8%), making it the second most common CAUTI uropathogen. Despite this frequent occurrence, the cellular and molecular details of C. albicans infection in the CAUTI microenvironment are poorly understood. Here, we characterize fungal virulence mechanisms and fungal biofilm formation during CAUTI for the first time. We found that the catheterized bladder environment triggers Candida virulence programs and robust biofilm formation through Efg1-dependent hyphal morphogenesis and Als1, an Efg1-downstream effector. Additionally, we show that the adhesin Als1 is necessary for in vitro and in vivo C. albicans biofilm formation dependent on the presence of fibrinogen (Fg), a coagulation factor released in the bladder due to the mechanical damage caused by urinary catheterization. Furthermore, in the presence of Fg, overexpression of ALS1 in C. albicans led to enhanced colonization and dissemination, while deletion of ALS1 reduced both outcomes during CAUTIs. Our study ultimately unveils the mechanism that contributes to fungal CAUTI, which may provide more effective targets for future therapies to prevent these infections.

Increased brain growth in escaped rainbow trout

Laberge, F. — 2021-06-18

Recent examples of rapid brain size plasticity in response to novel laboratory environments suggest that fish brain size is a flexible trait, allowing growth or shrinkage of brain tissue based on short term needs. Nevertheless, it remains to be seen if plasticity of fish brain size is relevant to natural environmental conditions. Here, using rainbow trout escaped from a farming operation as a natural experiment, we demonstrate that adult fish brain size can change rapidly in response to life in a natural lake environment. Specifically, escaped trout had on average 15% heavier brains relative to body size than captive trout after living for about 7 months in the lake. Because relative brain size of most escaped trout fell above the range of variation seen within the captive trout population, we conclude that increased brain size was achieved by plasticity after escape. Brain morphology analysis showed that the most anterior regions (olfactory bulbs and rest of telencephalon) contributed most to the increase in overall brain size in escaped trout. Relative size of the heart ventricle, another organ which can be subject to plastic changes under variable environmental conditions in fish, did not differ between escaped and captive trout. Massive and selective brain growth under the changed environmental conditions associated with escape from holding pens highlighted the plastic potential of fish brain size and suggests that a shift to increased complexity of life in the wild setting of a lake imposed greatly increased cognitive requirements on escaped trout.

Alcohol and Vapourized Nicotine Co-Exposure During Adolescence Contribute Differentially to Sex-Specific Behavioural Effects in Adulthood

Ruffolo, J. — 2021-06-24

IntroductionCo-occurrence of e-cigarette use and alcohol consumption during adolescence is frequent. However, little is known about their long-lasting effects when combined. Here, we examined whether adolescent co-exposure to alcohol drinking and vapourized nicotine would impact reward- and cognition-related behaviours in adult male and female rats during adulthood. MethodsFour groups of male and female Sprague Dawley rats (n=8-11/group/sex) received either nicotine (JUUL 5% nicotine pods) or vehicle vapour daily between postnatal days 30-46, while having continuous voluntary access to ethanol and water during this time in a two-bottle preference design. Upon reaching adulthood, rats underwent behavioural testing utilizing Pavlovian conditioned approach testing, fear conditioning and a two-bottle alcohol preference test. ResultsA sex-dependent effect was found in the two-bottle preference test in adulthood such that females had a higher intake and preference for alcohol compared to males regardless of adolescent exposure; both male and female adult rats had greater alcohol preference compared to adolescents. Male rats exposed to vapourized nicotine with or without alcohol drinking during adolescence exhibited altered reward-related learning in adulthood, evidenced by enhanced levels of sign-tracking behaviour. Male rats that drank alcohol with or without nicotine vapour in adolescence showed deficits in associative fear learning and memory as adults. In contrast, these effects were not seen in female rats exposed to alcohol and nicotine vapour during adolescence. ConclusionsThe present study provides evidence that co-exposure to alcohol and vapourized nicotine during adolescence in male, but not female, rats produces longterm changes in reward- and cognition-related behaviours. ImplicationsThese findings enhance our understanding of the effects of alcohol drinking and nicotine vapour exposure in adolescence. Moreover, they highlight potential sex differences that exist in the response to alcohol and nicotine vapour, underscoring the need for follow-up studies elucidating the neurobiological mechanisms that drive these sex differences, as well as the long-term effects of alcohol and nicotine vapour use.

Machine Learning based Genome-Wide Association Studies for Uncovering QTL Underlying Soybean Yield and its Components

Yoosefzadeh Najafabadi, M. — 2021-06-24

Genome-wide association study (GWAS) is currently one of the important approaches for discovering quantitative trait loci (QTL) associated with traits of interest. However, insufficient statistical power is the limiting factor in current conventional GWAS methods for characterizing quantitative traits, especially in narrow genetic bases plants such as soybean. In this study, we evaluated the potential use of machine learning (ML) algorithms such as support vector machine (SVR) and random forest (RF) in GWAS, compared with two conventional methods of mixed linear models (MLM) and fixed and random model circulating probability unification (FarmCPU), for identifying QTL associated with soybean yield components. In this study, important soybean yield component traits, including the number of reproductive nodes (RNP), non-reproductive nodes (NRNP), total nodes (NP), and total pods (PP) per plant along with yield and maturity were assessed using 227 soybean genotypes evaluated across four environments. Our results indicated SVR-mediated GWAS outperformed RF, MLM and FarmCPU in discovering the most relevant QTL associated with the traits, supported by the functional annotation of candidate gene analyses. This study for the first time demonstrated the potential benefit of using sophisticated mathematical approaches such as ML algorithms in GWAS for identifying QTL suitable for genomic-based breeding programs.

Pharmacokinetics and Central Accumulation of Delta-9-Tetrahydrocannabinol (THC) and its Bioactive Metabolites are Influenced by Route of Administration and Sex

Baglot, S. L. — 2021-07-03

Up to a third of North Americans over 16 years old report using cannabis in the prior month, most commonly through inhalation. Animal models that reflect human cannabis consumption are critical to study its impacts on brain and behaviour. Nevertheless, most animal studies to date examine effects of cannabis through injection of delta-9-tetrahydrocannabinol (THC; primary psychoactive component of cannabis). THC injections produce markedly different physiological and behavioural effects than inhalation, likely due to distinctive pharmacokinetics of each administration route. The current study directly examined if administration route (injection versus inhalation), with dosing being matched on peak THC blood levels, alters the metabolism of THC, and the central accumulation of THC and its metabolites over time. Adult male and female Sprague-Dawley rats received either a single intraperitoneal injection of THC (2.5 mg/kg) or a single (15 min) session of inhaled exposure to THC distillate (100 mg/mL) vapour. Blood and brains were collected at 15, 30, 60, 90 and 240 minutes post-exposure for analysis of THC and metabolites through mass spectrometry-liquid chromatography. Inhalation results in immediate hypothermia, whereas injection results in delayed hypothermia. Despite achieving comparable peak concentrations of blood THC in both groups, our results indicate higher initial brain THC concentration following inhalation, whereas injection resulted in dramatically higher 11-OH-THC concentrations, a potent THC metabolite, in blood and brain that increased over time. Our results provide evidence that THC and its metabolites exhibit different pharmacokinetic profiles following inhalation versus injection, which could have significant impacts for data interpretation and generalizability. Accordingly, we suggest that translational work in the realm of THC and cannabis strongly consider using inhalation models over those that employ injection. HighlightsO_LIBody temperature as well as blood and brain levels of THC and metabolites differ based on administration route C_LIO_LITHC inhalation results in immediate hypothermia, whereas THC injection results in delayed hypothermia C_LIO_LITHC inhalation results in higher initial brain THC levels than injection C_LIO_LITHC injection results in higher blood & brain 11-OH-THC levels than inhalation C_LIO_LITranslational cannabis work should strongly consider using inhalation over injection C_LI

Message in a Bottle, Metabarcoding Enables Biodiversity Comparisons Across Ecoregions

Steinke, D. — 2021-07-06

BackgroundTraditional biomonitoring approaches have delivered a basic understanding of biodiversity, but they cannot support the large-scale assessments required to manage and protect entire ecosystems. This study employed DNA metabarcoding to assess spatial and temporal variation in species richness and diversity in arthropod communities from 52 protected areas spanning three Canadian ecoregions. ResultsThis study revealed the presence of 26,263 arthropod species in the three ecoregions and indicated that at least another 3,000-5,000 await detection. Results further demonstrate that communities are more similar within than between ecoregions, even after controlling for geographical distance. Overall -diversity declined from east to west, reflecting a gradient in habitat disturbance. Shifts in species composition were high at every site with turnover greater than nestedness, suggesting the presence of many transient species. ConclusionsDifferences in species composition among their arthropod communities confirm that ecoregions are a useful synoptic for biogeographic patterns and for structuring conservation efforts. The present results also demonstrate that metabarcoding enables large-scale monitoring of shifts in species composition, making it possible to move beyond the biomass measurements that have been the key metric employed in prior efforts to track change in arthropod communities.

Gut-derived metabolites influence neurodevelopmental gene expression and Wnt signalling events in a germ-free zebrafish model

Rea, V. — 2021-07-06

Small molecule metabolites produced by the microbiome are known to be neuroactive and are capable of directly impacting the brain and central nervous system, yet there is little data on the contribution of these metabolites to the earliest stages of neural development and neural gene expression. Here, we explore the impact of rearing zebrafish embryos in the absence of microbes on early neural development as well as investigate whether any potential changes can be rescued with treatment of metabolites derived from the zebrafish gut microbiota. Overall, we did not observe any gross morphological changes between treatments but did observe a significant decrease in neural gene expression in embryos raised germ-free, which was rescued with the addition of zebrafish metabolites. Specifically, we identified 361 genes significantly down regulated in GF embryos compared to conventionally raised embryos via RNA-Seq analysis. Of these, 42 were rescued with the treatment of zebrafish gut-derived metabolites to GF embryos. Gene ontology analysis revealed that these genes are involved in prominent neurodevelopmental pathways including transcriptional regulation and Wnt signalling. Consistent with the ontology analysis, we found alterations in the development of Wnt dependent events which is rescued in the GF embryos treated with metabolites.

Implications of trade network structure and population dynamics for food security and equality

Fair, K. R. — 2021-07-10

Given trades importance to maintaining food security, it is crucial to understand the relationship between human population growth, land use, food supply, and trade. We develop a metapopulation model coupling human population dynamics to agricultural land use and food production in "patches" (regions and countries) connected via trade networks. Patches that import sparingly or fail to adjust their demand sharply in response to changes in food per capita experience food insecurity. They fall into a feedback loop between increasing population growth and decreasing food per capita, particularly if they are peripheral to the network. A displacement effect is also evident; patches that are more central and/or import more heavily preserve their natural land states. Their reliance on imports means other patches must expand their agricultural land. These results emphasize that strategies for improving food security and equality must account for the combined effects of network topology and patch-level characteristics.

HIF-dependent Neuropeptide Y Receptor Y1 and Y5 expression sensitizes hypoxic cells to NPY stimulation

Medeiros, P. J. — 2021-07-15

Neuropeptide Y (NPY) is an abundant neurohormone in the central and peripheral nervous system involved in feeding behavior, energy balance, nociception, and anxiety. Several NPY receptor (NPYR) subtypes display elevated expression in many cancers including in breast cancer where this is exploited for imaging and diagnosis. Here, we show that NPY1R and NPY5R mRNA abundance is induced by hypoxia in a Hypoxia Inducible Factor (HIF)-dependent manner in breast cancer cell lines MCF7 and MDA-MB-231. The HIFs bind to several genomic regions upstream of the NPY1R and NPY5R transcription start sites. The MAPK/ERK pathway is activated more rapidly upon NPY5R stimulation in hypoxic cells compared to normoxic cells. This pathway requires IGF1R activity in normoxia, but not in hypoxic cells where they display resistance to the radiosensitizer and IGF1R inhibitor AG1024. Hypoxic cells proliferate and migrate more when stimulated with NPY relative to normoxic cells, with a more robust response observed with a Y5-specific agonist. Our data suggest that hypoxia induced NPYRs render hypoxic cells more sensitive to NPY stimulation. Considering that breast tissue receives a constant supply of NPY, and hypoxia is a common feature of the tumor microenvironment, breast tumors are the perfect storm for hyperactive NPYR. This study not only highlights a new relationship between the HIFs and NPYR expression and activity, but may inform the use of chemotherapeutics targeting NPYRs and hypoxic cells.

Structural and functional characterization of ubiquitin variant inhibitors for the JAMM-family deubiquitinases STAMBP and STAMBPL1

Guo, Y. — 2021-07-17

Ubiquitination is one of the most crucial post-translational protein modifications involved in a myriad of biological pathways. This process is reversed by deubiquitinases (DUBs) that deconjugate the ubiquitin (Ub) moiety or poly-Ub chains from substrates. In the past decade, tremendous efforts have been focused on targeting DUBs for drug discovery. However, most chemical compounds with inhibitory activity for DUBs suffer from mild potency and low selectivity. To overcome these obstacles, we developed a phage display-based protein engineering strategy for generating Ub variant (UbV) inhibitors and have previously successfully applied it to the Ub-specific protease (USP) family of cysteine proteases. In this work, we leveraged the UbV platform to target STAMBP, a member of the JAB1/MPN/MOV34 (JAMM) metalloprotease family of DUB enzymes. We identified two UbVs (UbVSP.1 and UbVSP.3) that bind to STAMBP with high affinity but differ in their selectivity for the closely related paralog STAMBPL1. We determined the STAMBPL1-UbVSP.1 complex structure by X-ray crystallography, revealing hotspots of the tight JAMM-UbV interaction. Finally, we show that UbVSP.1 and UbVSP.3 are potent inhibitors of the STAMBP isopeptidase activity, far exceeding the reported small-molecule inhibitor BC-1471. This work demonstrates that UbV technology is suitable to develop tool molecules for metalloproteases. These tools can be used to understand the cellular function of JAMM family DUBs.

Evaluating 87Sr/86Sr isotope ratios and Sr mass fractions in otoliths of different European freshwater fish species as fishery management tool in an Alpine foreland with limited geological variability

Zitek, A. — 2021-07-25

The focus of this study was to assess the potential of otolith microchemistry as a fishery management tool for different European freshwater fish species in an Alpine foreland with a diverse range of different water bodies but low geological variation. 87Sr/86Sr isotope and Sr/Ca ratios in water samples from 26 habitat sites in a pre-alpine catchment region around lake Chiemsee, Germany, an important region for recreational and economic fisheries, were analysed. 87Sr/86Sr isotope ratios and the Sr mass fractions in otoliths of 246 fish out of 16 species were determined using (laser ablation) inductively coupled plasma mass spectrometry ((LA)-ICP-MS). Habitats could be discriminated into three distinct strontium isotope regions (SIGs) and seven clusters with characteristic 87Sr/86Sr isotope and Sr/Ca ratios. The direct comparison of 87Sr/86Sr isotope ratios in water and otolith samples allowed to identify fish that might have been a) migrating b) transferred from other water bodies or c) stocked from fish farms. Sr/Ca ratios in water and the Sr mass fraction in otoliths were highly correlated, although significant differences between species from the same environment could be documented. Sr mass fractions in sagittae of Perca fluviatilis were about 60 % of those in sagittae of Coregonus spp and of lapilli of roach Rutilus rutilus from the same habitats. Different partition factors for water to otolith Sr/Ca mass fractions were determined for different species. Discrimination of fish otoliths by 87Sr/86Sr isotope ratios and Sr mass fractions according to habitat clusters was possible with success rates ranging from 92 % to 100 % for cyprinids, European perch Perca fluviatilis, whitefish Coregonus spp. and European grayling Thymallus thymallus, and was 74 % for salmonids. Otolith microchemistry proved to have great potential to serve as a fishery management tool at smaller spatial scales such as in the studied Alpine foreland when considering the limited variation of 87Sr/86Sr isotope and Sr/Ca ratios, the type and spatial distribution of habitats, and the species and question under investigation. HighlightsO_LIOtolith microchemistry applied in in area with limited geological variability C_LIO_LIFish transferred, stocked or migrated were identified C_LIO_LIRegressions between Sr/Ca ratios in water predict Sr mass fractions in otoliths C_LIO_LISpecies specific Sr discrimination from water into otoliths C_LIO_LIEuropean freshwater fish species assigned to habitat clusters of origin C_LI

Kveik brewing yeasts demonstrate wide flexibility in beer fermentation temperature and flavour metabolite production and exhibit enhanced trehalose accumulation

Foster, B. — 2021-07-27

AO_SCPLOWBSTRACTC_SCPLOWTraditional Norwegian Farmhouse ale yeasts, also known as kveik, have captured the attention of the brewing community in recent years. Kveik were recently reported as fast fermenting thermo- and ethanol tolerant yeasts with the capacity to produce a variety of interesting flavour metabolites. They are a genetically distinct group of domesticated beer yeasts of admixed origin with one parent from the "Beer 1" clade and the other unknown. While kveik are known to ferment wort efficiently at warmer temperatures, its range of fermentation temperatures and corresponding flavour metabolites produced, remain uncharacterized. In addition, the characteristics responsible for its increased thermotolerance remain largely unknown. Here we demonstrate variation in kveik strains at a wide range of fermentation temperatures and show not all kveik strains are equal in fermentation performance, flavour metabolite production and stress tolerance. Furthermore, we uncovered an increased capacity of kveik strains to accumulate intracellular trehalose, which likely contributes to its increased thermo- and ethanol tolerances. Taken together our results present a clearer picture of the future opportunities presented by Norwegian kveik yeasts and offer further insight into their applications in brewing.

Evaluating the feasibility and efficacy of a dual-modality nanoparticle contrast agent (Nanotrast-CF800) for image-guided sentinel lymph node mapping in the oral cavity of healthy dogs

Wan, J. — 2021-07-26

A combination of pre- and intraoperative sentinel lymph node (SLN) mapping techniques has been suggested to optimize SLN detection. A novel liposomal nanoparticle, Nanotrast-CF800 (CF800), utilizes computed tomography lymphography (CTL) and near infrared fluorescence imaging (NIRF) for image-guided surgery and SLN mapping. This novel tracer agent has not been evaluated in companion animals. The objective of this study was to evaluate the feasibility and efficacy of CF800 for SLN mapping in the oral cavity of healthy dogs and to report any local adverse effects. Six healthy adult purpose-bred research dogs randomly received either 1 mL (group 1) or 2 mL (group 2) of CF800 injected into the submucosa at the level of the right canine maxillary tooth. CTL and percutaneous NIRF were performed at 1, 3, and 10 minutes, then 1, 2, 4, 7, and 10 days post-injection (p.i). Overall, both CTL and NIRF identified SLNs in all dogs. The overall peak mean contrast enhancement of the SLNs was 73.98 HU (range 63.45-86.27 HU) at 2 days p.i. Peak fluorescence of the SLN occurred at 1 day p.i. The agent was retained within the SLN for at least 7 days for CTL and 4 days for NIRF. No adverse effects were observed. Local administration of CF800 was simple and feasible for the detection of SLNs using CTL+NIRF in the head and neck of healthy dogs and was not associated with significant local adverse events.

Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM): a method for detection of antimicrobial resistance in environmental samples

Smith, S. D. — 2021-08-06

Effective monitoring of antibiotic resistance genes and their dissemination in environmental ecosystems has been hindered by the cost and efficiency of methods available for the task. We developed a method entitled the Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM), a system implementing high-throughput sequencing to simultaneously sequence thousands of antibiotic resistant genes representing a full-spectrum of antibiotic resistance classes commonly seen in environmental systems. In this study, we demonstrated DARTE-QM by screening 662 antibiotic resistance genes within environmental samples originated from manure, soil, and animal feces, in addition to a mock-community used as a control to test performance. DARTE-QM offers a new approach to studying antibiotic resistance in environmental microbiomes, showing advantages in efficiency and the ability to scale for many samples. This method provides a means of data acquisition that will alleviate the obstacles that many researchers in this area currently face.

Comparative analysis of machine learning and evolutionary optimization algorithms for precision tissue culture of Cannabis sativa: Prediction and validation of in vitro shoot growth and development based on the optimization of light and carbohydrate sources

Pepe, M. — 2021-08-10

Micropropagation techniques offer opportunity to proliferate, maintain, and study dynamic plant responses in highly controlled environments without confounding external influences, forming the basis for many biotechnological applications. With medicinal and recreational interests for Cannabis sativa L. growing, research related to the optimization of in vitro practices is needed to improve current methods while boosting our understanding of the underlying physiological processes. Unfortunately, due to the exorbitantly large array of factors influencing tissue culture, existing approaches to optimize in vitro methods are tedious and time-consuming. Therefore, there is great potential to use new computational methodologies for analysing data to develop improved protocols more efficiently. Here, we first tested the effects of light qualities using assorted combinations of Red, Blue, Far Red, and White spanning 0-100 mol/m2/s in combination with sucrose concentrations ranging from 1-6 % (w/v), totaling 66 treatments, on in vitro shoot growth, root development, number of nodes, shoot emergence, and canopy surface area. Collected data were then assessed using multilayer perceptron (MLP), generalized regression neural network (GRNN), and adaptive neuro-fuzzy inference system (ANFIS) to model and predict in vitro Cannabis growth and development. Based on the results, GRNN had better performance than MLP or ANFIS and was consequently selected to link different optimization algorithms (genetic algorithm, biogeography-based optimization, interior search algorithm, and symbiotic organisms search) for prediction of optimal light levels (quality/intensity) and sucrose concentration for various applications. Predictions of in vitro conditions to refine growth responses were subsequently tested in a validation experiment and data showed no significant differences between predicted optimized values and observed data. Thus, this study demonstrates the potential of machine learning and optimization algorithms to predict the most favourable light combinations and sucrose levels to elicit specific developmental responses. Based on these, recommendations of light and carbohydrate levels to promote specific developmental outcomes for in vitro Cannabis are suggested. Ultimately, this work showcases the importance of light quality and carbohydrate supply in directing plant development as well as the power of machine learning approaches to investigate complex interactions in plant tissue culture.

Local overfishing patterns have regional effects on health of coral, and economic transitions can promote its recovery

Milne, R. — 2021-08-15

Overfishing has the potential to severely disrupt coral reef ecosystems worldwide, while harvesting at more sustainable levels instead can boost fish yield without damaging reefs. The dispersal abilities of reef species mean that coral reefs form highly connected environments, and the viability of reef fish populations depends on spatially explicit processes such as the spillover effect and unauthorized harvesting inside marine protected areas. However, much of the literature on coral conservation and management has only examined overfishing on a local scale, without considering how different spatial patterns of fishing levels can affect reef health both locally and regionally. Here, we simulate a coupled human-environment model to determine how coral and herbivorous reef fish respond to overfishing across multiple spatial scales. We find that coral and reef fish react in opposite ways to habitat fragmentation driven by overfishing, and that a potential spillover effect from marine protected areas into overfished patches helps coral populations far less than it does reef fish. We also show that ongoing economic transitions from fishing to tourism have the potential to revive fish and coral populations over a relatively short timescale, and that large-scale reef recovery is possible even if these transitions only occur locally. Our results show the importance of considering spatial dynamics in marine conservation efforts, and demonstrate the ability of economic factors to cause regime shifts in human-environment systems.

In the Line of Fire: Debris Throwing by Wild Octopuses

Godfrey-Smith, P. — 2021-08-19

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

Limited evidence of a genetic basis for sex determination in the common creek chub, Semotilus atromaculatus

Meuser, A. V. — 2021-08-22

1Sexual reproduction is almost universal in the animal kingdom; therefore, each species must have a mechanism for designating sex as male or female. Fish especially have a wide range of sex determining systems. While incompatible systems are thought to increase reproductive isolation, interspecific hybridization is common among groups such as cyprinid minnows, thus, studies such as this can provide insight into hybridization and evolutionary diversification of this clade. In the present study, we aimed to identify a genetic basis for sex determination in the common creek chub (Semotilus atromaculatus) using genotyping-by-sequencing (GBS) data. No sex-associated markers were found by RADSex or a GWAS using GEMMA, however, our Weir and Cockerham locus-specific FST analysis and discriminant analysis of principal components revealed some genetic differentiation between the sexes at several loci. While no explicit sex determination mechanism has been yet discovered in creek chub, these loci are potential candidates for future studies. This study also highlights technical challenges involved in studying sex determination in species with extremely variable mechanisms.

Control of invasive Phragmites australis (European common reed) alters macroinvertebrate communities

Robichaud, C. D. — 2021-08-27

Wetland restoration often involves invasive plant species suppression to encourage the recovery of native-dominated vegetation communities. However, assessment of recovery is usually focused only on vegetation and the response of other critical wetland biota, such as macroinvertebrates, is seldom assessed. We characterized the aquatic, semi-aquatic, and terrestrial macroinvertebrate communities in remnant, uninvaded marsh to identify restoration targets and compared this to the communities in Phragmites australis-invaded marsh, and in formerly invaded marsh that was treated with the herbicide glyphosate in 2016 to simultaneously evaluate the effects of invasion and of invasive species suppression. We sampled invertebrates in 2017 and 2018 to track two years following herbicide treatment. The invertebrate community composition captured by the emergence traps was similar between P. australis and remnant marsh, suggesting invasion has little effect on macroinvertebrate community structure. There was also high concordance between the aquatic and emerging invertebrate communities in the invaded and uninvaded habitats. In contrast, herbicide-treated sites had a unique community composition, characterized by very high densities of Chironomidae (Diptera) and low taxa richness and evenness. Herbicide-treated sites also exhibited low concordance between the aquatic and emerging invertebrate communities, potentially attributable to the sparse emerging vegetation cover providing limited substrates for emergence. Herbicide-based invasive species control results in considerable changes to the macroinvertebrate community in freshwater marshes for at least two years after treatment, which may have consequences for aquatic food webs and species that rely on macroinvertebrates as prey.

Dual perspective proteomics infectome profiling discovers Salmonella type III secretion system effector functions in macrophages

Geddes-McAlister, J. — 2021-09-01

Intracellular bacterial pathogens have evolved sophisticated infection strategies, including the release and secretion of virulence factors to interfere with host cell functions and to perturb immune responses. For Salmonella enterica serovar Typhimurium (S. Typhimurium), the type III secretion systems encoded on Salmonella pathogenicity islands (SPI) 1 and 2 mediates invasion of the bacterium into innate immune cells and regulates bacterial replication and survival within the hostile environment of the host, respectively. Here, we explore the temporal and strain-specific dual perspective response of both the host and pathogen during cellular infection via quantitative proteomics. We report time- and pathogenicity island-specific expression and secretion of infection-associated proteins (i.e., SL1344_1263, SL1344_3112, SL1344_1563, and YnhG) and regulated immune response proteins in macrophage, including Cd86, Cd40, Casp4, C3, IL-1, and Cd69). Through intracellular macrophage and in vivo murine models of infection, we reveal a role in virulence for three of the bacterial proteins (SL1344_1263, SL1344_1563, and YnhG), defining their importance as novel T3SS effectors. We characterize the temporal intra- and extracellular production of the effectors and identify their interaction networks in host cells, representing inhibitory and stimulatory pathways mounted by invading bacterial pathogens. Author SummaryThe relationship between a host and pathogen is intricate, and regulation of the host immune response correlates with the progressive timing of infection and tailored responses to the pathogen. Relying on detection and quantification of protein-level changes using mass spectrometry-based proteomics, we explore the production of known and novel effectors by Salmonella during intracellular survival within macrophage. Our results portray a role for these effectors in bacterial virulence using an in vivo murine model of infection, and we define a dynamic network of interaction between the effectors and host proteins. These interactions reveal opportunity for drug design to disrupt modulation of the host by the invading bacterium as a new strategy for combatting infection. Our approach is dynamic and universal, with the power to alter therapeutic discovery against infectious diseases.

Predicting long pendant edges in model phylogenies, with applications to biodiversity and tree inference

Bocharov, S. — 2021-09-12

AO_SCPLOWBSTRACTC_SCPLOWIn the simplest phylogenetic diversification model (the pure-birth Yule process), lineages split independently at a constant rate {lambda} for time t. The length of a randomly chosen edge (either interior or pendant) in the resulting tree has an expected value that rapidly converges to [Formula] as t grows, and thus is essentially independent of t. However, the behaviour of the length L of the longest pendant edge reveals remarkably different behaviour: L converges to t/2 as the expected number of leaves grows. Extending this model to allow an extinction rate (where < {lambda}), we also establish a similar result for birth-death trees, except that t/2 is replaced by t/2 {middle dot} (1 - /{lambda}). This complete tree may contain subtrees that have died out before time t; for the reduced tree that just involves the leaves present at time t and their direct ancestors, the longest pendant edge length L again converges to t/2. Thus, there is likely to be at least one extant species whose associated pendant branch attaches to the tree approximately half-way back in time to the origin of the entire clade. We also briefly consider the length of the shortest edges. Our results are relevant to phylogenetic diversity indices in biodiversity conservation, and to quantifying the length of aligned sequences required to correctly infer a tree. We compare our theoretical results with simulations, and with the branch lengths from a recent phylogenetic tree of all mammals.

Kalium rhodopsins: Natural light-gated potassium channels

Govorunova, E. G. — 2021-09-17

We report a family of K+ channels, kalium channelrhodopsins (KCRs) from a fungus-like protist. Previously known potassium channels, widespread and mainly ligand- or voltage-gated, share a conserved pore-forming domain and K+-selectivity filter. KCRs differ in that they are light-gated and they have independently evolved an alternative K+ selectivity mechanism. The KCRs are potent, highly selective of K+ over Na+, and open in less than 1 millisecond following photoactivation. Their permeability ratio PK/PNa of [~] 20 make KCRs powerful hyperpolarizing tools that suppress excitable cell firing upon illumination, demonstrated here in mouse cortical neurons. KCRs enable specific optogenetic photocontrol of K+ gradients promising for the study and potential treatment of potassium channelopathies such as epilepsy, Parkinsons disease, and long-QT syndrome and other cardiac arrhythmias. One-Sentence SummaryPotassium-selective channelrhodopsins long-sought for optogenetic research and therapy of neurological and cardiac diseases.

small RNA can move long distances through plant vasculature to influence gene expression in shoot apical meristems

Minow, M. A. A. — 2021-09-20

In plants, small RNA (sRNA) can regulate gene expression via post transcriptional gene silencing (PTGS) or through RNA-directed DNA methylation (RdDM) leading to transcriptional gene silencing (TGS). sRNA is mobile throughout the plant, with movement occurring short distances from cell-to-cell as well as long distances through the vasculature via phloem trafficking. The range of long-distance sRNA mediated signaling from the vasculature to the shoot apical meristem (SAM) is not clear. To investigate this, two independent transgenic approaches were used to examine trafficking of phloem-expressed sRNA to the SAM in Arabidopsis thaliana. First, the phloem companion-cell specific promoter SUC2 was used to drive expression of an inverted repeat complementary to FLOWERING LOCUS D (FD), a flowering time regulator expressed exclusively in the SAM. In a separate experiment, the SUC2 promoter was used to express an artificial microRNA (aMiR) designed to target a synthetic CLAVATA3 (CLV3) target in the SAM stem cells. Both systems provide evidence of a phloem-to-SAM sRNA communication axis connecting distal regions of the plant to the stem cells of the SAM, which ultimately gives rise to all shoot tissues, including gametes. Thus, phloem-to-SAM sRNA movement defines an important link between sRNA expressed in distal regions of the plant and the growing shoot. Importantly, phloem-to-SAM sRNA trafficking may allow somatic sRNA to direct SAM RdDM, fixing transient sRNA expression events into stable epigenetic changes.

Tracing the invasion of a leaf-mining moth in the Palearctic through DNA barcoding of historical herbaria

Kirichenko, N. I. — 2021-10-09

Historical herbaria are valuable sources of data in invasion biology. Here we study the invasion history of the lime leaf-miner, Phyllonorycter issikii, by surveying over 15 thousand herbarium specimens of limes (Tilia spp.) collected in the Palearctic during last 253 years (1764-2016). The majority of herbarium specimens with the pests mines (89%) originated from East Asia (1859-2015), whereas remaining 11% of specimens with the mines came from Europe, European Russia and Western Siberia (1987-2015). These results support the hypothesis of a recent Ph. issikii invasion from Eastern to Western Palearctic. Single molecule real-time sequencing of the COI barcode region of 93 archival larvae and pupae (7-162 years old) dissected from the mines on historical herbaria allowed to distinguish between Ph. issikii and Ph. messaniella, a polyphagous species rarely feeding on Tilia, which mines were found in herbarium from Europe dated by 1915-1942. We discovered 25 haplotypes of Ph. issikii, of which 16 haplotypes were present solely in East Asia, and revealed wide distribution of the species in China. Six haplotypes shared between Eastern and Western Palearctic suggest the contribution of Ph. issikii populations from the Russian Far East, China and Japan to the westward invasion.

A live yeast supplementation to gestating ewes improves bioactive molecules composition in colostrum with no impact on its bacterial composition and beneficially affects immune status of the offspring

Duniere, L. — 2021-10-15

Colostrum quality is of paramount importance in the management of optimal ruminant growth and infectious disease prevention in early life. Live yeast supplementation effect during the last month of gestation was evaluated on ewes colostrum composition. Two groups of ewes (n=14) carrying twin lambs were constituted and twins were separated into groups (mothered or artificially-fed) 12h after birth. Nutrient, oligosaccharides (OS), IgG and lactoferrin concentrations were measured over 72h after lambing, and bacterial community was described in colostrum collected at parturition (T0). Immune passive transfer was evaluated through IgG measurement in lamb serum. In both groups, colostral nutrient, OS concentrations and IgG concentrations in colostrum and lamb serum decreased over time, (p < 0.01) except for lactose, which slightly increased (p < 0.001) and lactoferrin which remained stable. Bacterial population was stable over time with high relative abundances of Aerococcaceae, Corynebacteriaceae, Moraxellaceae and Staphylococcaceae in T0-colostrum. No effect of supplementation was observed in nutrient and lactoferrin concentrations. In supplemented ewes, colostral IgG level was higher at T0 and a higher level of serum IgG was observed in lambs born from supplemented mothers and artificially-fed, while no effect of supplementation was observed in the mothered lambs groups. Using a metabolomic approach, we showed that supplementation affected OS composition with significantly higher levels of colostral Neu-5Gc compounds up to 5h after birth. No effect of supplementation was observed on bacterial composition. Our data suggest that live yeast supplementation offsets the negative impact of early separation and incomplete colostrum feeding in neonate lambs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/464371v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@8c4e5borg.highwire.dtl.DTLVardef@16c6cf6org.highwire.dtl.DTLVardef@1658d42org.highwire.dtl.DTLVardef@1cf0fa_HPS_FORMAT_FIGEXP M_FIG C_FIG

The biological sink of atmospheric H2 is more sensitive to spatial variation of microbial diversity than N2O and CO2 emissions in an agroecosystem

Baril, X. — 2021-10-16

The relationship between soil microbial diversity and agroecosystem functioning is controversial due to the elevated diversity level and the functional redundancy of microorganisms. A field trial was established to test the hypothesis that enhanced crop diversity with the integration of winter cover crops (WCC) in a conventional maize-soy rotation promotes microbial diversity and the biological sink of H2 in soil, while reducing N2O emissions to the atmosphere. Vicia villosa (hairy vetch), Avena sativa (oat), and Raphanus sativus (Daikon radish) were cultivated alone or in combinations and flux measurements were performed throughout two subsequent growing seasons. Soil acted as a net sink for H2 and as a net source for CO2 and N2O. CO2 flux was the most sensitive to WCC whereas a significant spatial variation was observed for H2 flux with soil uptake rates observed in the most productive area two-fold greater than the baseline level. Sequencing and quantification of taxonomic and functional genes were integrated to explain variation in trace gas fluxes with compositional changes in soil microbial communities. Fungal communities were the most sensitive to WCC, but neither community abundance nor beta diversity were found to be indicative of fluxes. The alpha diversity of taxonomic and functional genes, expressed as the number of effective species, was integrated into composite variables extracted from multivariate analyses. Only the composite variable computed with the inverse Simpsons concentration index displayed a reproducible pattern throughout both growing seasons, with functional genes and bacterial 16S rRNA gene defining the two most contrasting gradients. The composite variable was decoupled from WCC treatment and explained 19-20% spatial variation of H2 fluxes. Sensitivity of the trace gas exchange process to soil properties at the local scale was inconsistent among H2, N2O and CO2, with the former being the most related to microbial diversity distribution pattern.

Expression of coffee florigen CaFT1 reveals a sustained floral induction window associated with asynchronous flowering in tropical perennials

Cardon, C. H. — 2021-11-04

The behavior of florigen(s) and environment-influenced regulatory pathways that control flowering in tropical perennials with complex phenological cycles is poorly understood. Understanding the mechanisms underlying this process is important for food production in the face of climate change. To explore this, homologs of Arabidopsis florigen FLOWERING LOCUS T (CaFT1) and environment-related regulators CONSTANS (CO), PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and FLOWERING LOCUS C (FLC) were isolated from Coffea sp. L. (Rubiaceae). Overexpression of CaFT1 in Arabidopsis showed typical early-flowering and yeast two hybrid studies indicated CaFT1 binding to bZIP floral regulator, FD, demonstrates that CaFT1 is a coffee orthologue of florigen. Expression of CaFT1 and floral regulators were evaluated over one year using three contrasting genotypes: two C. arabica and one C. canephora. All genotypes showed active CaFT1 transcription from February until October, indicating a potential window for floral induction. CaCO expression, as expected, varied over the day period and monthly with day length, whereas expression of temperature-responsive homologs, CaFLC and CaPIF4, did not correlate with temperature changes. Using coffee as a model, we suggest a continuum of floral induction that allows different starting points for floral activation, which explains developmental asynchronicity and prolonged anthesis events in tropical perennial species. HighlightCoffee florigen CaFT1 and related regulators revealed an extended floral induction window clarifying the asynchronicity and influence of environment for flowering in tropical perennial crops, providing perspectives to its control.

Correlation scan: identifying genomic regions that affect genetic correlations applied to fertility traits

Olasege, B. S. — 2021-11-05

Reproductive traits are often genetically correlated. Yet, we dont fully understand the complexities, synergism, or trade-offs between male and female fertility. Here, we introduce correlation scan, a novel framework for identifying the drivers or antagonizers of the genetic correlation between male and female fertility traits across the bovine genome. The identification of these regions facilitates the understanding of the complexity of these traits. Although the methodology was applied to cattle phenotypes, using high-density SNP genotypes, the general framework developed can be applied to any species or traits, and it can easily accommodate genome sequence data.

Allelopathic effects of Epichloë fungal endophytes: experiment and meta-analysis

Hager, H. A. — 2021-11-13

Host-specific Epichloe spp.endophytic fungal symbionts of pooid grasses that produce herbivore-deterring alkaloids and alter the grass hosts metabolite and protein profiles. Early observations suggested that Epichloe may have negative allelopathic effects on neighbouring plant species, particularly Trifolium spp. clovers, but subsequent allelopathy tests produced variable results. We examined two hypotheses: (1) Epichloe strains differ in allelopathic effect, and (2) Epichloe allelopathy negatively affects other plant species. We performed a greenhouse experiment using root exudates from Lolium perenne L. hosting different E. festucae var. lolii (Latch, M.J. Chr. & Samuels) C.W. Bacon & Schardl strains to compare their allelopathic effects on native legumes and forbs. We then used meta-analysis to examine the evidence to date for allelopathic effects of Epichloe endophytes. We found little difference in effect among E. festucae var. lolii strains and very little evidence for negative allelopathic effects of Epichloe in cool-season grasses across a range of methodologies, target plant species, and response measures. Negative allelopathic effects were detected only for root hair measures, which were from a single study. Positive effects on biomass were found for some experimental subgroups, including legumes. However, the majority of response variables showed no evidence for Epichloe allelopathy. Although there is currently little evidence for negative Epichloe allelopathic effects, our meta-analysis identified several research gaps. Experiments testing the functional belowground effects of Epichloe presence may help to determine its effects on non-host plant performance via plant-soil feedbacks.

Multi-marker metabarcoding resolves subtle variations in freshwater condition: Bioindicators, ecological traits, and trophic interactions

Robinson, C. V. — 2021-11-15

Freshwater systems are experiencing rapid biodiversity losses resulting from high rates of habitat degradation. Ecological condition is typically determined through identifying either macroinvertebrate or diatom bioindicator assemblages and comparing them to their known tolerance to stressors. These comparisons are typically conducted at family or genus levels depending on the availability of taxonomic keys and expertise for focal groups. The objective of this study was to test whether a more taxonomically comprehensive assessment of communities in benthic samples can provide a different perspective of ecological conditions. DNA metabarcoding was used to identify macroinvertebrates and diatoms from kick-net samples collected from sites with different habitat status. Sites with good condition were associated with higher beta diversity as well as slightly higher directed connectance and modularity indicating higher resilience compared with fair condition sites. Indicator value and correlation analyses used DNA metabarcoding data to detect 29 site condition indicator species consistent with known bioindicators and expected relative tolerances. DNA metabarcoding and trophic network analysis also recovered 11 keystone taxa. This study demonstrates the importance of taxonomic breadth across trophic levels for generating biotic data to study ecosystem status, with the potential to scale-up ecological assessments of freshwater condition, trophic stability, and resilience.

Polyethylene glycol (PEG) methods are superior to acidification for secondary concentration of Adenovirus and MS2 in water

McLellan, N. L. — 2021-11-20

Enteric viruses are a leading cause of waterborne illness worldwide and surveillance studies lack standardization in method selection. The most common and cost-effective approach to concentrating viruses from water samples involves virus adsorption and elution (VIRADEL) procedures, followed by secondary concentration. There is a lack of consistency in how secondary concentration methods are practiced and some methods may have better recovery for particular groups of viruses. Secondary concentration methods typically involve precipitation and the most common methods employ organic flocculation (OF) by acidification at a pH of 3.5, or precipitation by polyethylene glycol (PEG) in combination with the addition of NaCl. In this study, the recovery of coliphage MS2 using the plaque assay and human adenovirus strain 41 (HAdV41) using cell-culture and qPCR assays were evaluated by OF and PEG secondary concentration of spiked samples of wastewater, surface water, and groundwater. The recovery of MS2 and HAdV41 by PEG precipitation was significantly higher than that by OF (p<0.0001) when viruses were detected by culture based methods and marginally better when HAdV41 was enumerated by qPCR (p<0.019). The recovery of HAdV41 by qPCR ranged from 75.3% to 94.4% (n=36). The mean recovery of MS2 by OF was 4.4% (0.9%-7.7%; n=14) and ranged from 57.1% to 87.9% (n=28) for the PEG methods. The poor recovery of MS2 by OF was attributed to inactivation or poor stability at acidic conditions as MS2 were not recovered in the supernatant following OF and centrifugation. The inconsistency and lack of justification for method selection in many studies calls for a systematic study to inform guidance and standardization with respect to the application of concentration methods for various water types and viral pathogens. IMPORTANCEMS2 should not be used as a process control for methods involving acidification and culture-based detection. The dense floc produced by the PEG method may have contributed to higher recoveries as the pellet was more compact and stable than the loose pellet formed by OF. Standard methods for the detection of enteric viruses and surrogates that involve acidification could be modified with PEG precipitation to uphold virus recovery and minimize inactivation.

Positive feedback promotes terrestrial emergence behaviour in an amphibious fish

Tigert, L. R. — 2021-11-30

Major ecological transitions such as the invasion of land by aquatic vertebrates have been hypothesised to be facilitated by positive feedback between habitat choice and phenotypic plasticity. We tested whether aquatic hypoxia, emergence behaviour, and plastic changes in gill surface area could create such a positive feedback loop and drive an amphibious fish to spend increasing amounts of time out of water. We found that terrestrially acclimated amphibious mangrove rivulus Kryptolebias marmoratus were more sensitive to, and less tolerant of, aquatic hypoxia relative to water-acclimated fish, which are necessary trade-offs for positive feedback to occur. Next, we acclimated fish to normoxic or hypoxic water with the opportunity to emerge for 7d to test the predictions that fish in hypoxic conditions should regularly leave water, reduce gill surface area, and become less hypoxia tolerant. Consistent with these predictions, fish in severe hypoxia spent almost 50% of the time out of water and coverage of the gill lamellae by an inter-lamellar cell mass almost doubled. Hypoxia acclimated fish were also more sensitive to acute aquatic hypoxia (emergence at higher oxygen levels), and lost equilibrium faster in hypoxic water compared to control fish. Thus, we show that a positive feedback loop develops in amphibious fish where emergence behaviour begets further emergence behaviour, driven by gill remodelling which reduces aquatic respiratory function. Such a scenario may explain how amphibious behaviour has repeatedly evolved in fishes that occupy hypoxic aquatic habitats despite the associated challenges of life on land.

SARS-CoV-2 wildlife surveillance in Ontario and Quebec, Canada

Greenhorn, J. E. — 2021-12-03

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic, is capable of infecting a variety of wildlife species. Wildlife living in close contact with humans are at an increased risk of SARS-CoV-2 exposure and if infected have the potential to become a reservoir for the pathogen, making control and management more difficult. ObjectiveTo conduct SARS-CoV-2 surveillance in urban wildlife from Ontario and Quebec, Canada, increasing our knowledge of the epidemiology of the virus and our chances of detecting spillover from humans into wildlife. MethodsUsing a One Health approach, we leveraged activities of existing research, surveillance, and rehabilitation programs among multiple agencies to collect samples from 776 animals from 17 different wildlife species between June 2020 and May 2021. Samples from all animals were tested for the presence of SARS-CoV-2 viral RNA, and a subset of samples from 219 animals across 3 species (raccoons, Procyon lotor; striped skunks, Mephitis mephitis; and mink, Neovison vison) were also tested for the presence of neutralizing antibodies. ResultsNo evidence of SARS-CoV-2 viral RNA or neutralizing antibodies was detected in any of the tested samples. ConclusionAlthough we were unable to identify positive SARS-CoV-2 cases in wildlife, continued research and surveillance activities are critical to better understand the rapidly changing landscape of susceptible animal species. Collaboration between academic, public and animal health sectors should include experts from relevant fields to build coordinated surveillance and response capacity.

Reduction of free-roaming cat population requires high-intensity neutering in spatial contiguity to mitigate compensatory effects

Gunther, I. — 2021-12-03

When free-roaming in natural areas, the domestic cat (Felis silvestris catus) is ranked high among the most destructive alien species. Near human dwellings, it might risk humans, impair sanitation, and suffer from poor welfare. Cats popularity as companion animals complicates their population control. Thus, culling is often replaced by a fertility control method called "Trap-Neuter-Return/Release (TNR)," which is considered more humane. Despite the extensive application of TNR, a long-term controlled study was never performed to test its effectiveness. We present a uniquely designed controlled field experiment for examining TNR effectiveness. The study was performed over a twelve-year period, divided into pre-intervention, mixed- and full-intervention phases, and spanned a 20Km2 urban area. Trends of cat, intact-female, and kitten counts, cat reproduction, and carcass reports were compared among study phases and areas with different neutering intensities. The cat population increased during the first two study phases and did not decline in highly neutered populations, presumably due to cat immigration. Expansion of high-intensity neutering to the entire city in the full-intervention phase (>70% neutering percentage) reversed cat population growth, reaching an annual ca. 7% reduction. This population reduction was limited by a rebound increase in cat reproduction and longevity. We conclude that cat population management by TNR should be performed in high-intensity, continuously, and in geographic contiguity to enable population reduction. To enhance management effectiveness and mitigate compensatory effects, we recommend further evaluating an integrated strategy that combines TNR with complementary methods (e.g., vital resource regulation, ill cat euthanasia, and adoption). Significance StatementThough popular companion animals, domestic cats pose numerous problems when free-roaming, including predation of wildlife, hazards to humans, impaired sanitation, and a decrease in their welfare. Thus, managing their populations is essential. The Trap-Neuter-Return method (TNR, capturing, sterilizing, returning/releasing) is widely employed for managing cat populations. However, there is a lack of long-term controlled evidence for its effectiveness. We examined the outcomes of high-intensity TNR by performing a twelve-year controlled field experiment, the largest to date. Neutering over 70% of the cats caused population decline when applied over contiguous areas. However, it was limited by a rebound increase in reproduction and survival. These findings provide a robust quantification of the limitations and the long-term effectiveness of TNR.

Prevalence and Characteristics of Cannabis-induced Toxicoses in Pets: Results from a Survey of Veterinarians in North America

Khokhar, J. Y. — 2021-12-14

Cannabis legalization in North America has coincided with an increase in reports of cannabis-induced toxicosis in pets, but the magnitude of this problem, as well as outcomes of these incidents remain unknown. Therefore, we examined the frequency, diagnostic criteria, clinical signs, and prognoses of cannabis toxicoses in pets in North America. We conducted an online survey between January, 2021 and April, 2021 targeting veterinarians practicing in Canada and the United States (US). Out of the 251 study participants, 191 practiced in Canada. Cannabis toxicosis was most commonly reported in dogs (n=226 veterinarians), and the number of toxicosis cases increased significantly in Canada (p<0.0001) and the US (p=0.002) after October, 2018. Frequently reported clinical signs of cannabis toxicosis included: urinary incontinence (n=195), disorientation (n=182), ataxia (n=178), lethargy (n=150), hyperesthesia (n=134), and bradycardia (n=112). Edibles were most commonly suspected to be the cause of toxicosis (n=116). The most common route of exposure was ingestion (n=135), while the most cited reason was ingestion while unattended (n=135). Cannabis toxicosis was mostly diagnosed using supportive clinical signs (n=229), the most common treatment was outpatient monitoring (n=182), and pets were most often treated as out-patients (n=103). The legalization of cannabis use in Canada and the US is likely an important factor associated with the increased cannabis toxicosis cases in pets; however, the legal status may also increase reporting. The medicinal use of cannabis by pet-owners for pets may also contribute to a portion of the reported toxicoses. Most pets that experienced cannabis toxicosis recovered completely, suggesting that most cannabis toxicoses do not result in long-term ill effects. Even though some deaths (n=16) were reported in association with cannabis toxicosis, the presence of confounders such as toxins, and underlying conditions cannot be ruled out, emphasizing the need for rigorous controlled laboratory studies to investigate this important issue.

Signaling modality within gp130 receptor enhances tissue regeneration

Shkhyan, R. — 2022-01-05

Adult mammals are incapable of multi-tissue regeneration and augmentation of this potential may drastically shift current therapeutic paradigms. Here, we found that a common co-receptor of IL-6 cytokines, glycoprotein 130 (gp130), serves as a major nexus integrating various context-specific signaling inputs to either promote regenerative outcomes or aggravate disease progression. Via genetic and pharmacological experiments in vitro and in vivo, we demonstrated that a signaling tyrosine 814 (Y814) within gp130 serves as a major cellular stress sensor. Mice with constitutively inactivated Y814 (F814) exhibit regenerative, not reparative, responses after wounding in skin and anti-degenerative responses in the synovial joint. In addition, pharmacological inhibition of gp130 Y814 results in regeneration of multiple tissues in several species as well as disease modification in animal models of osteoarthritis. Our study characterizes a novel molecular mechanism that, if selectively manipulated, enhances the intrinsic regenerative capacity while preventing pathological outcomes in injury and disease. SummaryGp130 Y814 signaling module serves as a cellular stress sensor responsible for hindering tissue regeneration while triggering pathological outcomes after injury.

In vivo growth of Staphylococcus lugdunensis is facilitated by the concerted function of heme and non-heme iron acquisition mechanisms

Flannagan, R. S. — 2022-01-14

Acquisition of iron underpins the ability of pathogens to cause disease and Staphylococcus lugdunensis has increasingly been recognized as a pathogen that can cause serious infection. In this study, we sought to address the knowledge gap that exists regarding the iron acquisition mechanisms employed by S. lugdunensis, especially during infection of the mammalian host. Here we show that S. lugdunensis utilizes diverse genome encoded iron acquisition mechanisms to satisfy its need for this nutrient. Indeed, S. lugdunensis can usurp hydroxamate siderophores, and staphyloferrin A and B from S. aureus, using the fhuC ATPase-encoding gene. Acquisition of catechol siderophores and catecholamine stress hormones necessitates the presence of the sst-1 transporter-encoding locus, but not the sst-2 locus. Iron-dependent growth in acidic culture conditions necessitates the feoAB locus. Heme iron is acquired via expression of the iron-regulated surface determinant (isd) locus. During systemic infection of mice we demonstrate that while S. lugdunensis does not cause overt illness, it does colonize and proliferate to high numbers in the kidneys. By combining mutations in the various iron acquisition loci, we further demonstrate that only a strain mutated for all of isd, fhuC, sst-1, and feo, versus combination mutants carrying wild type copies of any one of those loci, was attenuated in its ability to proliferate to high numbers in kidneys. Taken together our data reveal that S. lugdunensis requires a repertoire of both heme and non-heme iron acquisition mechanisms to proliferate during systemic infection of mammals. ImportanceAcquisition of iron underpins the ability of pathogens to cause disease and Staphylococcus lugdunensis has increasingly been recognized as a pathogen that can cause serious infection. In this study, we sought to address the knowledge gap that exists regarding the iron acquisition mechanisms employed by S. lugdunensis, especially during infection of the mammalian host. Owing to an inability to synthesize siderophores, growth of S. lugdunensis is dramatically impaired in the presence of transferrin or serum, yet S. lugdunensis nonetheless uses several other genome-encoded iron acquisition mechanisms, in concert, to proliferate within the mammalian host. Therefore, the development of interventions that target bacterial iron acquisition systems should consider the overlapping function of distinct metal acquisition strategies deployed by bacterial pathogens.

SLDP and LIPA mediate lipid droplet-plasma membrane tethering in Arabidopsis thaliana

Krawczyk, H. E. — 2022-01-14

Membrane contact sites (MCS) are inter-organellar connections that allow for the direct exchange of molecules, such as lipids or Ca2+ between organelles, but can also serve to tether organelles at specific locations within cells. Here we identified and characterised three proteins that form a lipid droplet (LD)-plasma membrane (PM) tethering complex in plant cells, namely LD-localised SEED LD PROTEIN (SLDP) 1 and 2 and PM-localised LD-PLASMA MEMBRANE ADAPTOR (LIPA). Using proteomics and different protein-protein interaction assays, we show that both SLDPs associate with LIPA. Disruption of either SLDP1 and 2 expression, or that of LIPA, leads to an aberrant clustering of LDs in Arabidopsis seedlings. Ectopic co-expression of one of the SLDPs with LIPA on the other hand is sufficient to reconstitute LD-PM tethering in Nicotiana tabacum pollen tubes, a cell type characterised by dynamically moving LDs in the cytosolic streaming. Further, confocal laser scanning microscopy revealed both SLDP2.1 and LIPA to be enriched at LD-PM contact sites in seedlings. These and other results suggest that SLDP and LIPA interact to form a tethering complex that anchors a subset of LDs to the PM during post-germinative seedling growth in Arabidopsis thaliana. One-sentence summarySEED LIPID DROPLET PROTEIN1 and 2 and LIPID DROPLET PLASMA MEMBRANE ADAPTOR tether lipid droplets to the plasma membrane in seedlings of Arabidopsis thaliana.

Predator-prey interactions in a warming world: the critical role of cold tolerance

Ge, X. — 2022-01-20

Thermal tolerance mismatch within predator-prey systems may have pro-found effects on species population abundances and geographical distributions. To examine the generalized responses of a predator-prey system to climate change, we construct a biologically detailed stage-structured population dynamic model of interactions between ladybird beetles and aphids. We explore the models dynamics across the entire feasible parameter space of mean temperature and seasonality. Within this space, we explore different scenarios of predator and prey thermal tolerance mismatch to gain insight into how these thermal sensitivities affect the interacting species responses to climatic change. Our results indicate a predators cold tolerance has a larger effect on prey abundance than its heat tolerance. Mismatches between the predators and preys thermal tolerances also affect the species response to climate change. We identify three common patterns of species abundance across the feasible parameter space that relate to the type of thermal tolerance mismatches. Our study highlights the importance of understanding the complex interplay between climate change and species interactions.

Differential UBE2H-CTLH E2-E3 ubiquitylation modules regulate erythroid maturation

Sherpa, D. — 2022-01-18

The development of haematopoietic stem cells into mature erythrocytes - erythropoiesis - is a controlled process characterized by cellular reorganisation and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin (UB) system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the UB system is poorly understood. By measuring the proteomic landscape of in vitro human erythropoiesis models, we found dynamic differential expression of subunits of the CTLH E3 ubiquitin ligase complex that formed distinct maturation stage-dependent assemblies of structurally homologous RANBP9-and RANBP10-CTLH complexes. Moreover, protein abundance of CTLHs cognate E2-conjugating enzyme UBE2H increased during terminal differentiation, which depended on catalytically active CTLH E3 complexes. CRISPR-Cas9 mediated inactivation of all CTLH E3 assemblies by targeting the catalytic subunit MAEA, or UBE2H, triggered spontaneous and accelerated maturation of erythroid progenitor cells including increased heme and haemoglobin synthesis. Thus, the orderly progression of human erythropoiesis is controlled by the assembly of distinct UBE2H-CTLH modules functioning at different developmental stages.

Slower organisms exhibit sudden population disappearances in a reddened world

Greyson-Gaito, C. J. — 2022-01-28

Nature is replete with variation in the body sizes, reproductive output, and generation times of species that produce life history responses known to vary from small and fast to large and slow. Although researchers recognize that life history speed likely dictates fundamental processes in consumer-resource interactions like productivity and stability, theoretical work remains incomplete in this critical area. Here, we examine the role of life history speed on consumer-resource interactions by using a well used mathematical approach that manipulates the speed of the consumers growth rate in a consumer-resource interaction. Importantly, this approach holds the isocline geometry intact allowing us to assess the impacts of altered life history speed on stability (coefficient of variation, CV) without changing the underlying qualitative dynamics. Although slowing life history can be initially stabilizing, we find that in stochastic settings slowing ultimately drives highly destabilizing population disappearances, especially under reddened noise. Our results suggest that human-driven reddening of noise may decrease species stability because the autocorrelation of red noise enlarges the period and magnitude of perturbations, overwhelming a species natural compensatory responses via a ratchet-like effect. This ratchet-like effect then pushes species population dynamics far away from equilibria that can lead to precipitous local extinction.

Understanding Chicks' Emotions: Are Eye Blinks & Facial Temperatures Reliable Indicators?

Pijpers, N. — 2022-02-01

In commercial farming systems, chicks are reared without a mother. This absence of maternal influence can cause welfare problems when the chicks become older. Chicks imprint on their mothers they are young, and this mediates their stress and fear response. It is important to recognise problems early in the development of chicks to avoid welfare issues when they are older. One way to assess welfare is by measuring affective states. Research has shown chickens can display empathy, both towards their offspring and towards conspecifics. Measures of negative and positive affective states, either behavioural or physiological, could be good welfare indicators. This study employed non-invasive methods to measure affective states in laying hen chicks. Using video and thermal imaging, it analysed temperature changes in the peripheral areas and head region as well as changes in blinking behaviour before and after exposure to a stressor. The prediction was that the temperature would decrease in the eye and peripheral regions in response to a stressor and that the blinking rate would decrease. These changes would be indicative of a negative affective state. The results showed that the eye temperature as well as the blinking rate both decreased, whereas the temperature in the head region and the beak area increased. These results could be indicative of a negative affective state.

Interference of Neuronal TrkB Signaling by the Cannabis-Derived Flavonoids Cannflavins A and B

Holborn, J. — 2022-02-06

Cannflavins A and B are flavonoids that accumulate in the Cannabis sativa plant. These specialized metabolites are uniquely prenylated and highly lipophilic, which, a priori, may permit their interaction with membrane-bound enzymes and receptors. Although previous studies found that cannflavins can produce anti-inflammatory responses by inhibiting the biosynthesis of pro- inflammatory mediators, the full extent of their cellular influence remains to be understood. Here, we studied these flavonoids in relation to the Tropomyosin receptor kinase B (TrkB), a receptor tyrosine kinase that is activated by the growth factor brain-derived neurotrophic factor (BDNF). Using mouse primary cortical neurons, we first collected evidence that cannflavins prevent the accumulation of Activity-regulated cytoskeleton-associated (Arc, also known as Arg3.1) protein upon TrkB stimulation by exogenous BDNF in these cells. Consistent with this effect, we also observed a reduced activation of TrkB and downstream signaling effectors that mediate Arc mRNA transcription when BDNF was co-applied with the cannflavins. Of note, we also performed a high-throughput screen that demonstrated a lack of agonist action of cannflavins towards 320 different G protein-coupled receptors, a result that specifically limit the possibility of a TrkB transinactivation scenario via G protein signaling to explain our results with dissociated neurons. Finally, we used Neuro2a cells overexpressing TrkB to show that cannflavins can block the growth of neurites and increased survival rate produced by the higher abundance of the receptor in this model. Taken together, our study offers a new path to understand the reported effects of cannflavins and other closely related compounds in different cellular contexts.

Human Cerebral Spheroids Undergo Activity Dependent Changes In Cellular Composition And MicroRNA Expression

Parmentier, T. — 2022-02-06

Activity-induced neurogenesis has been extensively studied in rodents but the lack of ante mortem accessibility to human brain at the cellular and molecular levels limits studies of the process in humans. Using cerebral spheroids derived from human induced pluripotent stem cells (iPSCs), we investigated the effects of increased neuronal activity on neurogenesis. Our studies demonstrate that increasing neuronal activity with 4-aminopyridine in 3-month-old cerebral spheroids is associated with increases in the numbers of new neurons and decreases in the population of new glial cells. We also observed a significant decrease in the expression of miR-135a, which has previously been shown to be decreased in exercise-induced neurogenesis. Predicted targets of miR-135a include key participants in the SMAD2/3 and BDNF pathways. Together, our results suggest that iPSC-derived cerebral spheroids are an attractive model to study some aspects of activity-induced neurogenesis.

An increase in serial sarcomere number induced via weighted downhill running improves work loop performance in the rat soleus

Hinks, A. — 2022-02-20

Increased serial sarcomere number (SSN) has been observed in rats via downhill running training due to the emphasis on active lengthening contractions; however, little is known about the influence on dynamic contractile function. Therefore, we employed 4 weeks of weighted downhill running training in rats, then assessed soleus SSN and work loop performance. We hypothesized trained rats would produce greater net work output during faster, higher-strain work loops due to a greater SSN. Thirty-one Sprague-Dawley rats were assigned to a control or training group. Weight was added during downhill running via a custom-made vest, progressing from 5-15% body mass. Following sacrifice, the soleus was dissected, and a force-length relationship was constructed. Work loops (active shortening followed by passive lengthening) were then performed about optimal muscle length (LO) at 1.5-3-Hz cycle frequencies and 1-7-mm strains to assess net work output. Muscles were then fixed in formalin at LO. Fascicle lengths and sarcomere lengths were measured and used to calculate SSN. Intramuscular collagen content and crosslinking were quantified via a hydroxyproline content and pepsin-solubility assay. Trained rats had longer fascicle lengths (+13%), greater SSN (+8%), greater specific active forces (+50%), and lower passive forces (-45-62%) than controls (P<0.05). There were no differences in collagen parameters (P>0.05). Net work output was greater (+101-424%) in trained than control rats for the 1.5-Hz loops at 1, 3, and 5-mm strains (P<0.05) and showed relationships with fascicle length (R2=0.14-0.24, P<0.05). These results suggest training-induced longitudinal muscle growth may improve dynamic performance.

Highly divergent white-tailed deer SARS-CoV-2 with potential deer-to-human transmission

Pickering, B. — 2022-02-25

Wildlife reservoirs of SARS-CoV-2 may enable viral adaptation and spillback from animals to humans. In North America, there is evidence of unsustained spillover of SARS-CoV-2 from humans to white-tailed deer (Odocoileus virginianus), but no evidence of transmission from deer to humans. Through a biosurveillance program in Ontario, Canada we identified a new and highly divergent lineage of SARS-CoV-2 in white-tailed deer. This lineage is the most divergent SARS-CoV-2 lineage identified to date, with 76 consensus mutations (including 37 previously associated with non-human animal hosts) and signatures of considerable evolution and transmission within wildlife. Phylogenetic analysis also revealed an epidemiologically linked human case. Together, our findings represent the first clear evidence of sustained evolution of SARS-CoV-2 in white-tailed deer and of deer-to-human transmission.

Hidden diversity - DNA metabarcoding reveals hyper-diverse benthic invertebrate communities

Gleason, J. E. — 2022-03-02

Freshwater ecosystems, such as streams, are facing increasing pressures from agricultural land use. Aquatic insects and other macroinvertebrates have historically been used as indicators of ecological condition and water quality in freshwater biomonitoring programs; however, many of these protocols use coarse taxonomic resolution (e.g., family) when identifying macroinvertebrates. The use of family-level identification can mask species-level diversity, as well as patterns in community composition in response to environmental variables. Recent literature stresses the importance of robust biomonitoring to detect trends in insect decline globally, though most of these studies are carried out in terrestrial habitats. Here, we incorporate molecular identification (DNA metabarcoding) into a stream biomonitoring sampling design to explore the diversity and variability of aquatic macroinvertebrate communities at small spatial scales. We sampled twenty southern Ontario streams in an agricultural landscape for aquatic macroinvertebrates and, using DNA metabarcoding, revealed incredibly rich benthic communities which were largely comprised of rare taxa detected only once per stream despite multiple biological replicates. In addition to numerous rare taxa, our species pool estimates indicated that after 240 samples from twenty streams, there was a large proportion of taxa present which remained undetected by our sampling regime. When comparing different levels of taxonomic resolution, we observed that using OTUs revealed over ten times more taxa than family-level identification. A single insect family, the Chironomidae, contained over one third of the total number of OTUs detected in our study. Within-stream dissimilarity estimates were consistently high for all taxonomic groups (invertebrate families, invertebrate OTUs, chironomid OTUs), indicating stream communities are very dissimilar at small spatial scales. While we predicted that increased land use would homogenize benthic communities, this was not supported as within-stream dissimilarity was unrelated to land use.

Cytologic Scoring of Equine Exercise-Induced Pulmonary Hemorrhage (EIPH): Performance of Human Experts and a Deep Learning-Based Algorithm

Bertram, C. A. — 2022-03-02

Exercise-induced pulmonary hemorrhage (EIPH) is a relevant respiratory disease in sport horses which can be diagnosed by examination of bronchoalveolar lavage fluid (BALF) cells using the total hemosiderin score (THS). The aim of this study was to evaluate the diagnostic accuracy and reproducibility of trained annotators and to validate a deep learning-based algorithm for the THS. Digitized, iron-stained cytological specimens were prepared from 52 equine BALF samples. Ten annotators produced a THS for each slide according to published methods. The reference methods for comparing annotators and algorithmic performance included a ground truth dataset, the mean annotators THSs, and chemical iron measurements. Results of the study showed that annotators had marked inter-observer variability of the THS, which was mostly due to a systematic error between annotators in grading the intracytoplasmatic hemosiderin content of individual macrophages. Regarding overall measurement error between the annotators, 87.7% of the variance of the could be reduced by using standardized grades based on the ground truth. The algorithm was highly consistent with the ground truth in assigning hemosiderin grades. Compared to the ground truth THS, annotators had an accuracy of diagnosing EIPH (THS of < or [≥] 75) of 75.7% whereas the algorithm had an accuracy of 92.3% with no marked difference in correlation to chemical iron measurements. The results show that deep learning-based algorithms are useful for improving reproducibility and routine applicability of the THS. For THS by experts, a diagnostic uncertainty interval of 40 to 110 is proposed. THSs within this interval have insufficient reproducibility regarding the EIPH diagnosis.

PCYT2 controls muscle health and muscle aging

Cikes, D. — 2022-03-03

Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing, affecting hundreds of millions of people. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases represents an important goal in improving human health. Here, we show that phosphatidylethanolamine cytidyltransferase (PCYT2/ECT), the critical enzyme of the Kennedy branch of phosphatidylethanolamine (PE) synthesis pathway, has an essential role in muscle health. Human genetic deficiency in PCYT2 causes a severe disease with failure to thrive and progressive muscle weakness. Pcyt2 mutant zebrafish recapitulate the patient phenotypes, indicating that the role of PCYT2/PE in muscle is evolutionary conserved. Muscle specific Pcyt2 knockout mice exhibited failure to thrive, impaired muscle development, progressive muscle weakness, muscle loss, accelerated ageing, and reduced lifespan. Mechanistically, Pcyt2 deficiency affects mitochondrial bioenergetics and physicochemical properties of the myofiber membrane lipid bilayer, in particular under exercise strain. We also show that PCYT2 activity declines in the aging muscles of humans and mice. AAV-based delivery of PCYT2 rescued muscle weakness in Pcyt2 knock-out mice and, importantly, improved muscle strength in old mice, offering a novel therapeutic avenue for rare disease patients and muscle aging. Thus, PCYT2 plays a fundamental, specific, and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2 synthesized PE lipids to severe muscle dystrophy, exercise intolerance and aging.

Maternal immune activation and adolescent alcohol exposure increases alcohol drinking and disrupts cortical-striatal-hippocampal oscillations in adult offspring

Henricks, A. M. — 2022-03-04

Maternal immune activation (MIA) is strongly associated with an increased risk of developing mental illness in adulthood, which often co-occurs with alcohol misuse. The current study aimed to begin to determine whether MIA, combined with adolescent alcohol exposure (AE), could be used as a model with which we could study the neurobiological mechanisms behind such co-occurring disorders. Pregnant Sprague-Dawley rats were treated with PolyI:C or saline on gestational day 15. Half of the offspring were given continuous access to alcohol during adolescence, leading to four experimental groups: controls, MIA, AE, and Dual (MIA + AE). We then evaluated whether MIA and/or AE alters: 1) alcohol consumption; and 2) cortical-striatal-hippocampal oscillations in adult offspring. Dual rats, particularly females, drank significantly more alcohol in adulthood compared to all other groups. Using machine learning to build predictive models from oscillations, we were able to differentiate Dual rats from control rats and AE rats in both sexes, and Dual rats from MIA rats in females. The current data suggest that MIA+AE (Dual "hits") is a valuable model that we can use to study the neurobiological underpinnings of co-occurring disorders. Our future work aims to extend these findings to other addictive substances to enhance the translational relevance of this model.

Towards a multi-stressor theory for coral reefs in a changing world

Bieg, C. — 2022-03-25

Coral reefs are facing a constant barrage of human impacts, including eutrophication, overharvesting and climate change. While the local effects of overharvesting have been well-studied, regional nutrient loading from anthropogenic activities on land and global climate change-induced disturbances are increasing in magnitude and necessitating cross-scale multi-stressor approaches for coral reef ecology. Here, we expand on longstanding theory to develop an integrated multi-stressor framework for coral reefs. We show that: i) the geometry of a simple, empirically-motivated model suggests nutrients and harvesting can operate similarly, and synergistically, in driving shifts from coral- to algae-dominated reefs, resulting in clear context-dependent management implications; and ii) this same geometry suggests climate-driven coral mortality can drive the presence of long transients and climate-driven alternate states, even in moderately-impacted ecosystems. Reefs seemingly in a "safe space" based on individual stressors may in fact be much more susceptible to increasingly frequent storms and bleaching events in multi-stressor conditions. By integrating these findings with general ecological and theoretical concepts, we suggest that responses in benthic composition may act as "signatures of change" to multi-stressors, allowing us to develop a predictive and generalizable multi-stressor framework for coral reefs under global change. In line with this theory, we detail empirical evidence from Barbados of historical changes in reef composition and multi-stressor impacts within our framework. By bridging coral reef ecology and general ecological concepts, we can better understand ecosystem functioning and resilience in these important yet highly threatened systems. Manuscript HighlightsO_LITheoretical understanding of synergistic multi-stressor impacts on coral reefs C_LIO_LIUnexpected climate-driven alternate states, related to long transients C_LIO_LITheoretical framework predicts "signatures of change" based on dominant stressor C_LI

Phylogenomics Illuminates the Evolutionary History of Wild Silkmoths in Space and Time (Lepidoptera: Saturniidae)

Rougerie, R. — 2022-03-30

AO_SCPLOWBSTRACTC_SCPLOWWild silkmoths (Saturniidae) are one of the most emblematic and most studied families of moths. Yet, the absence of a robust phylogenetic framework based on a comprehensive taxonomic sampling impedes our understanding of their evolutionary history. We analyzed 1,024 ultraconserved elements (UCEs) and their flanking regions to infer the relationships among 338 species of Saturniidae representing all described subfamilies, tribes, and genera. We investigated systematic biases in genomic data and performed dating and historical biogeographic analyses to reconstruct the evolutionary history of wild silkmoths in space and time. Using Gene Genealogy Interrogation, we showed that saturation of nucleotide sequence data blurred our understanding of early divergences and first biogeographic events. Our analyses support a Neotropical origin of saturniids, shortly after the Cretaceous-Paleogene extinction event (ca 64.0 [stem] - 52.0 [crown] Ma), and two independent colonization events of the Old World during the Eocene, presumably through the Bering Land Bridge. Early divergences strongly shaped the distribution of extant subfamilies as they showed very limited mobility across biogeographical regions, except for Saturniinae, a subfamily now present on all continents but Antarctica. Overall, our results provide a framework for in-depth investigations into the spatial and temporal dynamics of all saturniid lineages and for the integration of their evolutionary history into further global studies of biodiversity and conservation. Rather unexpectedly for a taxonomically well-known family such as Saturniidae, the proper alignment of taxonomic divisions and ranks with our phylogenetic results leads us to propose substantial rearrangements of the family classification, including the description of one new subfamily and two new tribes.

Decision Tree Ensembles Utilizing Multivariate Splits Are Effective at Investigating Beta-Diversity in Medically Relevant 16S Amplicon Sequencing Data

Rudar, J. — 2022-04-01

Developing an understanding of how microbial communities vary across conditions is an important analytical step. We used 16S rRNA data isolated from human stool to investigate if learned dissimilarities, such as those produced using unsupervised decision tree ensembles, can be used to improve the analysis of the composition of bacterial communities in patients suffering from Crohns Disease and adenomas/colorectal cancers. We also introduce a workflow capable of learning dissimilarities, projecting them into a lower dimensional space, and identifying features that impact the location of samples in the projections. For example, when used with the centered log-ratio transformation, our new workflow (TreeOrdination) could identify differences in the microbial communities of Crohns Disease patients and healthy controls. Further investigation of our models elucidated the global impact ASVs had on the location of samples in the projected space and how each ASV impacted individual samples in this space. Furthermore, this approach can be used to integrate patient data easily into the model and results in models that generalize well to unseen data. Models employing multivariate splits can improve the analysis of complex high-throughput sequencing datasets since they are better able to learn about the underlying structure of the dataset. Author SummaryThere is an ever-increasing level of interest in accurately modeling and understanding the role that commensal organisms play in human health and disease. We show that learned representations can be used to create informative ordinations. We also demonstrate that the application of modern model introspection algorithms can be used to investigate and quantify the impact of taxa in these ordinations and that the taxa identified by these approaches have been associated with immune-mediated inflammatory diseases and colorectal cancer.

Development of a long-read NGS workflow for improved characterization of fastidious respiratory mycoplasmas

Framst, I. — 2022-04-01

Mycoplasmas are respiratory pathogens in humans and animals and due to their fastidious nature, they have been historically underdiagnosed. Lack of standardised diagnostic, typing and antimicrobial susceptibility testing methods makes clinical management and epidemiological studies challenging. The aim of this study was to develop a cost-effective and accurate sequencing workflow for genotypic characterization of clinical isolates of respiratory mycoplasmas using a rapid long-read sequencing platform. Critical aspects of bacterial whole genome sequencing were explored using fastidious respiratory Mycoplasma (M. felis and M. cynos) isolated from animals including: (i) four solid and liquid-based media based on a specialized formulation for Mycoplasma culture, (ii) three DNA extraction methods modified for sequencing purposes, and (iii) two de novo assembly platforms as key components of a bioinformatic pipeline including Flye and Canu assemblers. DNA quality and quantity compatible with long-read sequencing requirements were obtained with culture volumes of 160ml in modified Hayflicks broth incubated for 96 hours. The other three culture approaches investigated did not meet the DNA quality criteria required for long-read sequencing. The use of bead-beating bacterial cell lysis in the extraction protocol resulted in smaller fragments and shorter reads compared to enzymatic lysis methods. Overall, Flye generated more contiguous assemblies than the Canu assembler. This novel study provides a step-by-step sequencing workflow including mycoplasma culture, DNA extraction and de novo assembly approaches for the characterization of highly fastidious respiratory mycoplasmas. This workflow will provide diagnosticians, epidemiologists, and researchers with a more comprehensive tool than the laborious conventional methods for a complete genomic characterization of respiratory mycoplasmas.

Multi-marker DNA metabarcoding detects suites of environmental gradients from an urban harbour

Robinson, C. V. — 2022-04-18

There is increasing need for biodiversity monitoring, especially in places where potential anthropogenic disturbance may significantly impact ecosystem health. We employed a combination of traditional morphological and bulk macroinvertebrate metabarcoding analyses to benthic samples collected from Toronto Harbour (Ontario, Canada) to compare taxonomic and functional diversity of macroinvertebrates and their responses to environmental gradients. At the species rank, sites assessed using COI metabarcoding showed more variation than sites assessed using morphological methods. Depending on the assessment method, we detected gradients in magnesium (morphological taxa), ammonia (morphological taxa, COI sequence variants), pH (18S sequence variants) as well as gradients in contaminants such as metals (COI & 18S sequence variants) and organochlorines (COI sequence variants). Observed responses to contaminants such as aromatic hydrocarbons and metals align with known patchy distributions in harbour sediments. We determined that the morphological approach may limit the detection of macroinvertebrate responses to lake environmental conditions due to the effort needed to obtain fine level taxonomic assignments necessary to investigate responses. DNA metabarcoding, however, need not be limited to macroinvertebrates, can be automated, and taxonomic assignments are associated with a certain level of accuracy from sequence variants to named taxonomic groups. The capacity to detect change using a scalable approach such as metabarcoding is critical for addressing challenges associated with biodiversity monitoring and ecological investigations.

Route of oxytetracycline administration differentially impacts the growth and gut microbiome of pigs co-infected with Bordetella bronchiseptica and Pasteurella multocida

Mou, K. — 2022-04-19

Along with judicious antibiotic use, there is great interest in how the dose regimen of an antibiotic affects the animal gut microbiota. This study evaluated the impact of experimental respiratory infection alone or respiratory infection followed by oxytetracycline (oxytet) treatment on the animals health and its fecal microbiome. Piglets of approximately three weeks-of-age were separated into four groups (n=20 per group). One group remained non-infected and administered non-medicated feed and the other three groups were infected with Bordetella bronchiseptica (day 0) and Pasteurella multocida (day 4), with one group receiving non- medicated feed and the remaining two groups receiving oxytetr starting on day 7 by injection or in-feed (day 7-14). Infection with B. bronchiseptica and P. multocida negatively impacted piglet growth and induced mild pneumonia. Infection alone had minimal effect on the fecal microbiota community. When oxytet was administered either by injection or in-feed to treat the respiratory infections, both routes had minimal effect on clearing B. bronchiseptica and P. multocida in the animal. However, both routes appeared to limit lung lesion severity, and injected oxytet reduced the negative impact of infection on weight gain. Both routes had limited impact on the animals overall gut microbiome, including relative abundances of bacterial taxa and antibiotic resistance genes tet32, tetW, and aph2. Overall, oxytet administered by either route did not clear the respiratory infection, but oxytet administration minimized the negative health impacts of infection and had minor impact on the pig gut microbiome. ImportanceEfforts to address antibiotic resistance calls for improved antibiotic stewardship, including considering antibiotic administration route. While our previous study found in-feed oxytet had greater impact on the gut microbiome of healthy piglets than injected oxytet, it remained unknown if oxytet treatments would have the same impact on the microbiota of infected piglets. We evaluated the impact of respiratory infection alone or respiratory infection followed by oxytet treatment on the animals health and their gut microbiome profile. Respiratory infection negatively affected piglets health, but infection alone had minimal impact on the gut community. When oxytet was administered either in-feed or by injection to treat the respiratory infection, neither route of administration led to the clearance of the respiratory pathogens. However, oxytet minimized the negative health impacts of infection, and had minor impact on the pig gut microbiome. These findings are informative for disease management in food animals while integrating antibiotic stewardship practices.

A revised perspective on the evolution of troponin I and troponin T in vertebrates

Joyce, W. — 2022-04-22

The troponin (Tn) complex, responsible for the Ca2+ activation of striated muscle, is composed of three interacting protein subunits: TnC, TnI, and TnT, encoded by TNNC, TNNI, and TNNT genes. TNNI and TNNT are sister gene families, and in mammals the three TNNI paralogs (TNNI1, TNNI2, TNNI3), which encode proteins with tissue-specific expression, are each in close genomic proximity with one of the three TNNT paralogs (TNNT2, TNNT3, TNNT1, respectively). It has been widely presumed that all vertebrates broadly possess genes of these same three classes, although earlier work has overlooked jawless fishes (cyclostomes) and cartilaginous fishes (chimaeras, rays and sharks), which are distantly related to other jawed vertebrates. With a new phylogenetic and synteny analysis of a diverse array of vertebrates including these taxonomic groups, we define five distinct TNNI classes (TNNI1-5), with TNNI4 and TNNI5 being only present in non-mammalian vertebrates and typically found in tandem, and four classes of TNNT (TNNT1-4). These genes are located in four genomic loci that were generated by the 2R whole-genome duplication events. TNNI3, encoding cardiac TnI in mammals, was independently lost in cartilaginous and ray-finned fishes. Ray-finned fishes predominantly express TNNI1 in the heart. TNNI5 is highly expressed in shark hearts and contains an N-terminal extension similar to that of TNNI3 found in tetrapod hearts. Given that TNNI3 and TNNI5 are distantly related, this supports the hypothesis that the N-terminal extension may be an ancestral feature of vertebrate TNNI and not an innovation unique to TNNI3, as has been commonly believed.

Bacterial lipopolysaccharide modulates immune response in the colorectal tumor microenvironment

Sulit, A. K. — 2022-04-27

Immune responses can have opposing effects in colorectal cancer (CRC), the balance of which may determine whether a cancer regresses, progresses, or potentially metastasizes. These effects are evident in CRC consensus molecular subtypes (CMS) where both CMS1 and CMS4 contain immune infiltrates yet have opposing prognoses. The microbiome has previously been associated with CRC and immune response in CRC but has largely been ignored in the CRC subtype discussion. Using CMS subtyping, we aimed to determine the contributions of the microbiome to the pleiotropic effects evident in immune-infiltrated subtypes. We integrated host gene-expression and meta-transcriptomic data to determine the link between immune characteristics and microbiome contributions in these subtypes and identified lipopolysaccharide (LPS) binding as a potential functional mechanism. We identified candidate bacteria with LPS properties that could affect immune response, focusing on Fusobacterium periodonticum and Bacteroides fragilis in CMS1, and Porphyromonas asaccharolytica in CMS4. Treatment of peripheral blood mononuclear cells (PBMCs) with LPS isolated from these bacteria showed that F. periodonticum stimulates cytokine production in PBMCs while both B. fragilis and P. asaccharolytica had an inhibitory effect. Furthermore, LPS from the latter two species can inhibit the immunogenic properties of F. periodonticum LPS when co-incubated with PBMCs. We propose that different microbes in the CRC tumor microenvironment can alter the local immune activity, with important implications for prognosis and treatment response.

StandardRat: A multi-center consensus protocol to enhance functional connectivity specificity in the rat brain

Grandjean, J. — 2022-04-28

Task-free functional connectivity in animal models provides an experimental framework to examine connectivity phenomena under controlled conditions and allows comparison with invasive or terminal procedures. To date, animal acquisitions are performed with varying protocols and analyses that hamper result comparison and integration. We introduce StandardRat, a consensus rat functional MRI acquisition protocol tested across 20 centers. To develop this protocol with optimized acquisition and processing parameters, we initially aggregated 65 functional imaging datasets acquired in rats from 46 centers. We developed a reproducible pipeline for the analysis of rat data acquired with diverse protocols and determined experimental and processing parameters associated with a more robust functional connectivity detection. We show that the standardized protocol enhances biologically plausible functional connectivity patterns, relative to pre-existing acquisitions. The protocol and processing pipeline described here are openly shared with the neuroimaging community to promote interoperability and cooperation towards tackling the most important challenges in neuroscience.

Metabarcoding metacommunities: time, space, and land use interact to structure aquatic macroinvertebrate communities in streams

Gleason, J. E. — 2022-05-01

There is an increasing need to move beyond evaluating the effect of land use on stream communities by only studying local variables, and instead incorporate a metacommunity perspective which integrates environmental and spatial factors across larger spatial scales. The use of molecular tools (DNA metabarcoding) to identify bioindicator groups, such as aquatic macroinvertebrates, can provide greater taxonomic resolution to explore patterns in stream metacommunities. In this study, we collected aquatic macroinvertebrates from streams in southern Ontario which spanned a gradient of agricultural disturbance and used DNA metabarcoding to identify the species composition from these samples. We address a significant knowledge gap in previous stream aquatic macroinvertebrate metacommunity studies by incorporating molecular identification as well as a temporal component. We observed that a combination of local habitat conditions, regional agricultural land use, and spatial position influenced aquatic macroinvertebrate community composition, suggesting there is an interaction between environmental filtering and dispersal processes that structures these communities. However, aquatic macroinvertebrate communities were also highly dissimilar between streams and composed of many rare species, and a large percentage of unexplained variation suggests that there is a strong stochastic component to community assembly. We also observed that there is a seasonal component to metacommunity dynamics, with different water quality variables being significant to community composition in each sampling month. While we expected that an increased percentage of surrounding agricultural land use would result in more homogenous macroinvertebrate communities, we only detected this relationship in May and found evidence that a larger riparian buffer width can mitigate the effects of agricultural land use. We demonstrate the utility of DNA metabarcoding for revealing patterns in metacommunity dynamics that may not be detectable using coarse taxonomic identifications, and reveal the importance of incorporating a seasonal component when evaluating the influence of land use on community composition.

Genetic and pharmacological reduction of CDK14 mitigates synucleinopathy

Parmasad, J.-L. A. — 2022-05-02

Parkinsonas disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of -Synuclein (-Syn) protein. Currently, no treatment can slow nor halt the progression of PD. Multiplications and mutations of the -Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress -Syn replicate several features of PD. Decreasing total -Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of -Syn levels and identified CDK14, a kinase of largely unknown function as a regulator of -Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we ablated Cdk14 in the -Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical -Syn pathology, indicated by reduced numbers of pS129 -Syn-containing cells. In primary neurons, we found that Cdk14 depletion protects against the propagation of toxic -Syn species. We further validated these findings on pS129 -Syn levels in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated -Syn in human neurons, in PFF- challenged rat neurons and in the brains of -Syn-humanized mice. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.

A real data-based simulation procedure to select an imputation strategy for mixed-type trait data

May, J. A. — 2022-05-04

Missing observations in trait datasets pose an obstacle for analyses in myriad biological disciplines. Considering the mixed results of imputation, the wide variety of available methods, and the varied structure of real trait datasets, a framework for selecting a suitable imputation method is advantageous. We invoked a real data-driven simulation strategy to select an imputation method for a given mixed-type (categorical, count, continuous) target dataset. Candidate methods included mean/mode imputation, k-nearest neighbour, random forests, and multivariate imputation by chained equations (MICE). Using a trait dataset of squamates (lizards and amphisbaenians; order: Squamata) as a target dataset, a complete-case dataset consisting of species with nearly complete information was formed for the imputation method selection. Missing data were induced by removing values from this dataset under different missingness mechanisms: missing completely at random (MCAR), missing at random (MAR), and missing not at random (MNAR). For each method, combinations with and without phylogenetic information from single gene (nuclear and mitochondrial) or multigene trees were used to impute the missing values for five numerical and two categorical traits. The performances of the methods were evaluated under each missing mechanism by determining the mean squared error and proportion falsely classified rates for numerical and categorical traits, respectively. A random forest method supplemented with a nuclear-derived phylogeny resulted in the lowest error rates for the majority of traits, and this method was used to impute missing values in the original dataset. Data with imputed values better reflected the characteristics and distributions of the original data compared to complete-case data. However, caution should be taken when imputing trait data as phylogeny did not always improve performance for every trait and in every scenario. Ultimately, these results support the use of a real data-driven simulation strategy for selecting a suitable imputation method for a given mixed-type trait dataset. Author summaryThe issue of missing data is problematic in trait datasets as the missingness pattern may not be entirely random. Whether data are missing may depend on other known observations in the dataset, or on the value of the missing data points themselves. When only complete cases are used in an analysis, derived results may be biased. Imputation is an alternative to complete-case analysis and entails filling in the missing values using information provided by other trait values present in the dataset. Including phylogenetic information in the imputation process can improve the accuracy of imputed values, though results are dependent on the amount and pattern of missingness. Most previous evaluations of imputation methods for trait datasets are limited to numerical simulated data, with categorical traits not considered. Given a particular dataset, we propose the use of a real data-driven simulation strategy to select an imputation method. We evaluated the accuracies of four different imputation methods, with and without phylogeny information, and under different simulated missingness patterns using an example reptile trait dataset. Results indicated that data imputed using the best-performing method better reflected the original dataset characteristics compared to complete-case data. As imputation performance varies depending on the properties of a given dataset, a real data-driven simulation strategy can be used to provide guidance on best imputation practices.

Identification of sex-determining loci in hybridizing Catostomus fish species

Pyne, C. B. — 2022-05-04

1Despite the near-universality of gonochorism (separate sexes) in eukaryotic organisms, the underlying mechanisms of sex determination are poorly understood and highly variable in some taxa. Sex determination mechanisms may promote or impede reproductive isolation depending on whether mechanisms are similar between related species, so identifying genomic regions associated with sex determination is especially relevant for understanding evolutionary diversification and hybridization between closely related species. In Catostomus fishes, contemporary hybridization is variable and extensive, but factors influencing hybridization dynamics are not fully understood. In the present study, we aim to describe the genetic basis of sex determination in bluehead (Catostomus discobolus) and white suckers (Catostomus commersonii ) to understand the potential impact of sex determination on reproductive isolation. We used genotyping-by-sequencing data from Catostomus species and their hybrids to identify regions of the genome associated with sex using a genome-wide association study and the identification of sex-specific loci. We identified a genetic basis of sex determination in Catostomus fishes, with a region of the genome significantly associating with sex in bluehead suckers. This region is suggestive of a master sex-determining region in bluehead suckers but is not significant in white suckers, implying that either the sex-determining region of the genome differs in these two species that hybridize, or that sample size was insufficient to identify this genomic region in white suckers. By describing and comparing sex-determination systems across Catostomus fish species, we highlight the relationship between sex determining systems and hybridization in closely related fish species.

Quantifying exposure of bumblebee (Bombus spp.) queens to pesticide residues when hibernating in agricultural soils

Rondeau, S. — 2022-05-11

Exposure to pesticides is a major threat to bumblebee (Bombus spp.) health. In temperate regions, queens of many bumblebee species hibernate underground for several months, putting them at potentially high risk of exposure to soil contaminants. The extent to which bumblebees are exposed to residues in agricultural soils during hibernation is currently unknown, which limits our understanding of the full pesticide exposome for bumblebees throughout their lifecycle. To generate field exposure estimates for overwintering bumblebee queens to pesticide residues, we sampled soils from areas corresponding to suitable likely hibernation sites at six apple orchards and 13 diversified farms throughout Southern Ontario (Canada) in fall 2019-2020. Detectable levels of pesticides were found in 65 of 66 soil samples analysed for multi-pesticide residues (UPLC-MS/MS). A total of 53 active ingredients (AIs) were detected in soils, including 27 fungicides, 13 insecticides, and 13 herbicides. Overall, the frequency of detection, residue levels (median = 37.8 vs. 2.2 ng/g), and number of pesticides per sample (mean = 12.3 vs. 4.3 AIs) were highest for orchard soils compared to soils from diversified farms. Ninety-one percent of samples contained multiple residues, including mixtures of insecticides and fungicides that might lead to synergistic effects. Up to 29 different AIs were detected per soil sample. Our results suggest that when hibernating in agricultural areas, bumblebee queens are very likely to be exposed to a wide range of pesticide residues in soil. Our study indicates the importance of empirically testing the potential effects of pesticide residues in soils for hibernating bumblebee queens, using field exposure data such as those generated here. The differences in exposure that we detected between cropping systems can also be used to better inform regulations that govern the use of agricultural pesticides, notably in apple orchards.

Cysteine Restriction-Specific Effects of Sulfur Amino Acid Restriction on Lipid Metabolism

Nichenametla, S. N. — 2022-05-15

Decreasing dietary intake of methionine exerts robust anti-adiposity effects in rodents but modest effects in humans. Since cysteine can be synthesized from methionine, animal diets are formulated by decreasing methionine and eliminating cysteine. Such diets exert both methionine restriction (MR) and cysteine restriction (CR), i.e., sulfur amino acid restriction (SAAR). Contrarily, human diets used in clinical studies did not eliminate cysteine and thus might have exerted only MR. Epidemiological studies positively correlate body adiposity with plasma cysteine but not with methionine, suggesting that CR, but not MR, is responsible for the antiadiposity effects of SAAR in rodents. Whether this is true, and if so, the underlying mechanisms are unknown. Using multiple diets with variable concentrations of methionine and cysteine, we demonstrate that the anti-adiposity effects of SAAR are due to CR. CR increased serinogenesis (serine biosynthesis from non-glucose substrates) by diverting substrates from glyceroneogenesis, essential for fatty acid/triglyceride cycling. Molecular data suggest that the CR results in glutathione depletion, which induces Nrf2 and downstream targets Phgdh (the serine biosynthetic enzyme) and Pepck-M. Using multiple mouse models, we show that the magnitude of SAAR-induced changes in molecular markers depends on dietary fat concentration (60 fat>10% fat), gender (males>females), and age-at-onset (young>adult). Our findings are translationally relevant as we found a negative correlation of plasma serine with triglycerides and metabolic syndrome criteria in a cross-sectional epidemiological study. SAAR-like diets with high polyunsaturated fatty acids increased plasma serine in a short-term human feeding study. Serinogenesis might be a potential target to correct hypertriglyceridemia.

Vertical and horizontal transmission of cell fusing agent virus in Aedes aegypti

Logan, R. A. — 2022-05-26

Cell fusing agent virus (CFAV) is an insect specific flavivirus (ISF) found in field and laboratory populations of Aedes aegypti. ISFs have recently demonstrated the ability to block the transmission of arboviruses such as dengue, West Nile and Zika viruses. It is thought that vertical transmission is the main route for ISF infections. This has been observed with CFAV, but there is evidence of horizontal and venereal transmission in other ISFs. Understanding the route of transmission can inform strategies to spread ISFs to wild vector populations as a method of controlling pathogenic arboviruses. We crossed individually reared male and female mosquitoes from both a naturally occurring CFAV-positive Ae. aegypti colony and its negative counterpart to provide information on maternal, paternal, and horizontal transmission. RT-PCR was used to detect CFAV in individual female mosquito pupal exuviae and was 89% sensitive, but only 41% in male mosquito pupal exuviae. This is a possible way to screen individuals for infection without destroying the adults. Female-to-male horizontal transmission was not observed during this study, however there was a 31% transmission rate from mating pairs of CFAV-positive males to negative female mosquitoes. Maternal vertical transmission was observed with a filial infection rate of 93%. The rate of paternal transmission was 85% when the female remained negative, 61% when the female acquired CFAV horizontally, and 76% overall. Maternal and paternal transmission of CFAV could allow the introduction of this virus into wild Ae. aegypti populations through male or female mosquito releases, and thus provides a potential strategy for ISF-derived arbovirus control.

The ER protein translocation channel subunit Sbh1 controls virulence of Cryptococcus neoformans

Santiago-Tirado, F. H. — 2022-06-01

The fungal pathogen Cryptococcus neoformans is distinguished by a cell wall-anchored polysaccharide capsule that is critical for virulence. Biogenesis of both cell wall and capsule relies on the secretory pathway. Protein secretion begins with polypeptide translocation across the endoplasmic reticulum (ER) membrane through a highly conserved channel formed by three proteins: Sec61, Sbh1, and Sss1. Sbh1, the most divergent, contains multiple phosphorylation sites, which may allow it to regulate entry into the secretory pathway in a species- and protein-specific manner. Absence of SBH1 causes a cell-wall defect in both Saccharomyces cerevisiae and C. neoformans, although other phenotypes differ. Notably, proteomic analysis showed that when cryptococci are grown in conditions that mimic aspects of the mammalian host environment (tissue culture medium, 37 {degrees}C, 5% CO2), a set of secretory and transmembrane proteins is upregulated in wild-type, but not in{Delta} sbh1 mutant cells. The Sbh1-dependent proteins show specific features of their ER targeting sequences that likely cause them to transit less efficiently into the secretory pathway. Many also act in cell-wall biogenesis, while several are known virulence factors; consistent with these observations, the C. neoformans {Delta}sbh1 mutant is avirulent in a mouse infection model. We conclude that, in the context of conditions encountered during infection, Sbh1 controls the entry of virulence factors into the secretory pathway of C. neoformans, and thereby regulates fungal pathogenicity. ImportanceCryptococcus neoformans is a yeast that causes almost 200,000 deaths worldwide each year, mainly of immunocompromised individuals. The surface structures of this pathogen, a protective cell wall surrounded by a polysaccharide capsule, are made and maintained by proteins that are synthesized inside the cell and travel outwards through the secretory pathway. A protein called Sbh1 is part of the machinery that determines which polypeptides enter this export pathway. We found that when Sbh1 is absent, both C. neoformans and the model yeast S. cerevisiae show cell wall defects. Lack of Sbh1 also changes the pattern of secretion of both transmembrane and soluble proteins, in a manner that depends on characteristics of their sequences. Notably, multiple proteins that are normally upregulated in conditions similar to those encountered during infection, including several needed for cryptococcal virulence, are no longer increased. Sbh1 thereby regulates the ability of this important pathogen to cause disease.

Metagenomics vs. total RNA sequencing: most accurate data-processing tools, microbial identification accuracy, and implications for freshwater assessments

Hempel, C. A. — 2022-06-03

Metagenomics and total RNA sequencing (total RNA-Seq) have the potential to improve the taxonomic identification of diverse microbial communities, which could allow for the incorporation of microbes into routine freshwater assessments. However, these targeted-PCR- free techniques require more testing and optimization. In this study, we processed metagenomics and total RNA-Seq data from a commercially available microbial mock community using 768 data-processing workflows, identified the most accurate data-processing tools, and compared their microbial identification accuracy at equal and increasing sequencing depths. The accuracy of data-processing tools substantially varied among replicates. Total RNA-Seq was more accurate than metagenomics at equal sequencing depths and even at sequencing depths almost one order of magnitude lower than those of metagenomics. We show that while data-processing tools require further exploration, total RNA-Seq might be a favorable alternative to metagenomics for targeted-PCR-free taxonomic identifications of microbial communities and might enable a substantial reduction in sequencing costs while maintaining accuracy. This could be particularly an advantage for routine freshwater assessments, which require cost-effective yet accurate methods, and might allow for the incorporation of microbes into freshwater assessments.

Lake Erie fish safe to eat yet afflicted by algal hepatotoxins

Shahmohamadloo, R. S. — 2022-06-09

Microcystin toxins from harmful algal blooms (HABs) can accumulate and persist in fish, raising dual concerns about human health risks from consumption and the potential for detrimental impacts on fish populations. However, there are fundamental unknowns about the relationship between HABs and fish populations driven by a lack of field information on toxin accumulation and retention over space and time. We conducted a field study to assess human health risks from consuming fish caught across all life stages of a HAB and to determine the pervasiveness of potentially harmful levels of microcystins on fish populations. We collected 190 fish in 2015 and 2017 from Lake Erie, a large freshwater ecosystem that is highly productive for fisheries and is an epicenter of HABs and microcystin toxicity events. Muscles and livers were analyzed for total microcystins, which was used to conduct a human health risk assessment for comparison against fish consumption advisory benchmarks available for Lake Erie. We find low human health risk from muscle consumption following the World Health Organizations safety thresholds. However, all fish across capture dates had microcystins in their livers at levels shown to cause adverse effects, suggesting a pervasive and underappreciated toxic stressor. These data demonstrate that microcystins are retained in fish livers well beyond the cessation of HABs and calls for additional research to better understand the effects of sublethal toxic exposures for fish population dynamics, conservation, and related ecosystem services.

Zoonotic avian influenza viruses evade human BTN3A3 restriction

Pinto, R. M. — 2022-06-15

Cross-species transmission of avian influenza A viruses (IAVs) into humans could represent the first step of a future pandemic1. Multiple factors limiting the spillover and adaptation of avian IAVs in humans have been identified, but they are not sufficient to explain which virus lineages are more likely to cross the species barrier1,2. Here, we identified human BTN3A33 (butyrophilin subfamily 3 member A3) as a potent inhibitor of avian but not human IAVs. We determined that BTN3A3 is constitutively expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts at the early stages of virus replication by inhibiting avian IAV vRNA transcription. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F, or rarely 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, several serotypes of avian IAVs that spilled over into humans in recent decades evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure4. Importantly, we identified more than 150 avian IAV lineages with a BTN3A3-resistant genotype. In conclusion, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

Serotyping Salmonella Enteritidis and Typhimurium Using Whole Cell Matrix Assisted Laser Desorption Ionization - Time of Flight Mass Spectrometry (MALDI-TOF MS) through Multivariate Analysis and Artificial Intelligence

Gao, A. — 2022-07-02

Salmonella is one of the most frequent food-borne zoonoses, while Salmonella Typhimurium and Enteritidis are the major serovars of concern in public health. 113 Salmonella strains including 38 S. Enteritidis (SE), 38 S. Typhimurium (ST) and 37 strains of 32 other Salmonella serovars (SG) were tested in quadruplicate by whole-cell MALDI-TOF MS. Ions were studied and aligned from the raw data of mzXML files using Mass-Up (http://www.sing-group.org/mass-up), resulting in 1,741 aligned peaks. Datasets of ions (presence/absence) selected using a home-developed criteria on their specificity and detectability were subjected to multivariate analyses and artificial intelligence tools. Principle Component Analysis based on 88 selected ions separated SE, ST and SG without overlap on the first three principle components. The network and forest based deep learning tools were more sophisticated than the decision tree-based models. Neural Network carried out consistently well in training model, but no advantage was gained over the other models in validation results. HP (high performance) Neural, Support Vector Machine, HP Forest and Gradient Boasting were able to identify SE, ST and SG up to 100% correctly in both training and validation when 88 selected ions were used in analysis. Among them, HP Neural seemed to perform slightly better and relatively stable. Selection of serovar specific ions helps develop serotyping by increasing signal to noise. MALDI-TOF MS used with appropriate data processing strategies and classification tools could be applied to quickly alert when Salmonella serotypes of concern are suspected among routinely processed samples.

Phenotypic extremes or extreme phenotypes? On the use of large and small-bodied "phenocopied" Drosophila melanogaster males in studies of sexual selection and conflict

Schang, K. — 2022-07-28

In the fruit fly, Drosophila melanogaster, variation in body size is influenced by a number of different factors and may be strongly associated with individual condition, performance and success in reproductive competitions. Consequently, intra-sexual variation in size in this model species has been frequently explored in order to better understand how sexual selection and sexual conflict may operate and shape evolutionary trajectories. However, measuring individual flies can often be logistically complicated and inefficient, which can result in limited sample sizes. Instead, many experiments use large and/or small body sizes that are created by manipulating the developmental conditions experienced during the larval stages, resulting in "phenocopied" flies whose phenotypes resemble what is seen at the extremes of a populations size distribution. While this practice is fairly common, there has been remarkedly few direct tests to empirically compare the behaviour or performance of phenocopied flies to similarly-sized individuals that grew up under typical developmental conditions. Contrary to assumptions that phenocopied flies are reasonable approximations, we found that both large and small-bodied phenocopied males frequently differed from their standard development equivalents in their mating frequencies, their lifetime reproductive successes, and in their effects on the fecundity of the females they interacted with. Our results highlight the complicated contributions of environment and genotype to the expression of body size phenotypes and lead us to strongly urge caution in the interpretation of studies solely replying upon phenocopied individuals.

Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA

Maboni, G. — 2022-07-28

Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in clinical bacterial isolates. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria, we compared different approaches to identify best practices for detection of AMR genes, including: total genomic DNA and plasmid DNA extractions, solo assembly of Illumina short-reads and of ONT long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA indicating that, at least in these three enterobacterial genera, purification of plasmid DNA was not necessary to detect plasmid-borne AMR genes. We also found that Illumina short-reads used with ONT long-reads in either hybrid or polished assemblies of total genomic DNA enhanced sensitivity and accuracy of AMR gene detection. Phenotypic susceptibility corresponded well with genotypes identified by sequencing, but the three AMR databases differed significantly in distinguishing mobile dedicated AMR genes from non-mobile chromosomal housekeeping genes in which rare spontaneous resistance mutations might occur. This study reveals the need for standardized biochemical and informatic procedures and database resources for consistent, reliable AMR genotyping to take full advantage of WGS to expedite patient treatment and to track AMR genes within the hospital and community.

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

Gervais, N. C. — 2022-08-15

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPRa to overexpress genes involved in pathogenesis and drug resistance and contribute towards the identification of novel phenotypes. Together, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

Reconstruction of Small Subunit Ribosomal RNA from High-Throughput Sequencing Data: A Comparative Study of Metagenomics and Total RNA Sequencing

Hempel, C. A. — 2022-08-27

The small subunit (SSU) ribosomal RNA (rRNA) is the most commonly used marker for the identification of microbial taxa, but its full-length reconstruction from high-throughput sequencing (HTS) data remains challenging, especially for complex and diverse environmental samples. Metagenomics and total RNA sequencing (total RNA-Seq) are target-PCR-free HTS methods that are used to characterize microbial communities and simultaneously reconstruct SSU rRNA sequences. However, more testing is required to determine and improve their effectiveness. In this study, we processed metagenomics and total RNA-Seq data retrieved from a commercially available mock microbial community using 112 combinations of commonly used data-processing tools, determined SSU rRNA reconstruction completeness of both sequencing methods for each species in the mock community, and analyzed the impact of data-processing tools on SSU rRNA and genome completeness. Total RNA-Seq allowed for the complete or near-complete reconstruction of all mock community SSU rRNA sequences and outperformed metagenomics. SSU rRNA completeness of metagenomics strongly correlated with the genome size of mock community species. The impact of data-processing tools was overall low, although certain tools resulted in significantly lower SSU rRNA completeness. These results are promising for the high-throughput reconstruction of novel full-length SSU rRNA sequences and could advance the simultaneous application of multiple -omics approaches in routine environmental assessments to allow for more holistic assessments of ecosystems.

Low human health risks of algal toxins from consuming fish caught in Lake St. Clair

Shahmohamadloo, R. S. — 2022-09-12

Consuming fish exposed to cyanobacterial harmful algal blooms (HABs) may be a major route of microcystin toxin exposure to humans. However, it remains unknown whether fish can accumulate and retain microcystins temporally in waterbodies with recurring seasonal HABs, particularly before and after a HAB event when fishing is active. We conducted a field study on Largemouth Bass, Northern Pike, Smallmouth Bass, Rock Bass, Walleye, White Bass, and Yellow Perch to assess the human health risks to microcystin toxicity via fish consumption. We collected 124 fish in 2016 and 2018 from Lake St. Clair, a large freshwater ecosystem in the North American Great Lakes that is actively fished pre- and post-HAB periods. Muscles were analyzed using the MMPB Lemieux Oxidation method for total microcystins, which was used to perform a human health risk assessment for comparison against fish consumption advisory benchmarks available for Lake St. Clair. From this collection 35 fish livers were additionally extracted to confirm the presence of microcystins. Microcystins were detected in all livers at widely varying concentrations (1-1,500 ng g-1 ww), suggesting HABs are an underappreciated and pervasive stressor to fish populations. Conversely, microcystin levels were consistently low in muscles (0-15 ng g-1 ww) and presented negligible risk, empirically supporting that fillets may be safely consumed before and after HAB events following fish consumption advisories.

Nutrient addition increases grassland resistance during dry and wet climate extremes despite functional diversity loss

Chen, Q. — 2022-09-19

O_LINutrient enrichment impacts grassland plant diversity such as species richness, functional trait composition and diversity, but whether and how these changes affect ecosystem stability in the face of increasing climate extremes remains largely unknown. C_LIO_LIWe quantified the direct and diversity-mediated effects of nutrient addition (by nitrogen, phosphorus, and potassium) on the stability of aboveground biomass production in 10 long-term grassland experimental sites. We measured five facets of stability as the temporal invariability, resistance during and recovery after extreme dry and wet growing seasons. C_LIO_LILeaf traits (leaf carbon, nitrogen, phosphorus, potassium, and specific leaf area) were measured under ambient and nutrient addition conditions in the field and were used to construct the leaf economic spectrum (LES). We calculated functional trait composition and diversity of LES and of single leaf traits. We quantified the contribution of intraspecific trait shifts and species replacement to change in functional trait composition as responses to nutrient addition and its implications for ecosystem stability. C_LIO_LINutrient addition decreased functional trait diversity and drove grassland communities to the faster end of the LES primarily through intraspecific trait shifts, suggesting that intraspecific trait shifts should be included for accurately predicting ecosystem stability. Moreover, the change in functional trait diversity of the LES in turn influenced different facets of stability. That said, these diversity-mediated effects were overall weak and/or overwhelmed by the direct effects of nutrient addition on stability. As a result, nutrient addition did not strongly impact any of the stability facets. These results were generally consistent using individual leaf traits but the dominant pathways differed. Importantly, major influencing pathways differed using average trait values extracted from global trait databases (e.g. TRY). C_LIO_LISynthesis. Investigating changes in multiple facets of plant diversity and their impacts on multidimensional stability under global changes such as nutrient enrichment can improve our understanding of the processes and mechanisms maintaining ecosystem stability. C_LI

Audiovisual cues must be predictable and win-paired to drive risky choice

Hathaway, B. A. — 2022-09-22

Risky or maladaptive decision making is thought to be central to the etiology of both drug and gambling addiction. Salient audiovisual cues paired with rewarding outcomes, such as the jackpot sound on a win, can enhance disadvantageous, risky choice in both rats and humans, yet it is unclear which aspects of the cue-reward contingencies drive this effect. Here, we implemented six variants of the rat Gambling Task (rGT), in which animals can maximise their total sugar pellet profits by avoiding options paired with higher per-trial gains but disproportionately longer and more frequent time-out penalties. When audiovisual cues were delivered concurrently with wins, and scaled in salience with reward size, significantly more rats preferred the risky options as compared to the uncued rGT. Similar results were observed when the relationship between reward size and cue complexity was inverted, and when cues were delivered concurrently with all outcomes. Conversely, risky choice did not increase when cues occurred randomly on 50% of trials, and decision making actually improved when cues were coincident with losses alone. As such, cues do not increase risky choice by simply elevating arousal, or amplifying the difference between wins and losses. It is instead important that the cues are reliably associated with wins; presenting the cues on losing outcomes as well as wins does not diminish their ability to drive risky choice. Computational analyses indicate reductions in the impact of losses on decision making in all rGT variants in which win-paired cues increased risky choice. These results may help us understand how sensory stimulation can increase the addictive nature of gambling and gaming products.

Selection leads to remarkable variability in the outcomes of hybridization across replicate hybrid zones

McFarlane, S. E. — 2022-09-26

Hybrid zones have been viewed as an opportunity to see speciation in action. When hybrid zones are replicated, it is assumed that if the same genetic incompatibilities are maintaining reproductive isolation across all instances of secondary contact, those incompatibilities should be identifiable by consistent patterns in the genome. In contrast, changes in allele frequencies due to genetic drift should be idiosyncratic for each hybrid zone. To test this assumption, we simulated 20 replicates of each of 12 hybrid zone scenarios with varied genetic incompatibilities, rates of migration, selection and different starting population size ratios of parental species. We found remarkable variability in the outcomes of hybridization in replicate hybrid zones, particularly with Bateson-Dobzhansky-Muller incompatibilities and strong selection. We found substantial differences among replicates in the overall genomic composition of individuals, including admixture proportions, inter-specific ancestry complement, and number of ancestry junctions. Additionally, we found substantial variation in genomic clines among replicates at focal loci, regardless of locus-specific selection. We conclude that processes other than selection are responsible for some consistent outcomes of hybridization, whereas selection on incompatibilities can lead to genomically widespread and highly variable outcomes. We highlight the challenge of mapping between pattern and process in hybrid zones and call attention to how selection against incompatibilities will commonly lead to variable outcomes. We hope that this study informs future research on replicate hybrid zones and encourages further development of statistical techniques, theoretical models, and exploration of additional axes of variation to understand reproductive isolation.

Structural foundations of potassium selectivity in channelrhodopsins

Govorunova, E. G. — 2022-09-27

Kalium channelrhodopsins (KCRs) are light-gated K+ channels recently found in the stramenopile protist Hyphochytrium catenoides. When expressed in neurons, KCRs enable high-precision optical inhibition of spiking (optogenetic silencing). KCRs are capable of discriminating K+ from Na+ without the conventional K+-selectivity filter found in classical K+ channels. The genome of H. catenoides also encodes a third paralog that is more permeable for Na+ than for K+. To identify structural motifs responsible for the unusual K+ selectivity of KCRs, we systematically analyzed a series of chimeras and mutants of this protein. We found that mutations of three critical residues in the paralog convert its Na+ selective channel into a K+ selective one. Our characterization of homologous proteins from other protists (Colponema vietnamica, Cafeteria burkhardae and Chromera velia) and metagenomic samples confirmed the importance of these residues for K+ selectivity. We also show that Trp102 and Asp116, conserved in all three H. catenoides orthologs, are necessary, although not sufficient, for K+ selectivity. Our results provide the foundation for further engineering of KCRs for optogenetic needs. IMPORTANCERecently discovered microbial light-gated ion channels (channelrhodopsins) with a higher permeability for K+ than for Na+ (kalium channelrhodopsins, or KCRs) demonstrate an alternative K+ selectivity mechanism, unrelated to well-characterized "selectivity filters" of voltage- and ligand-gated K+ channels. KCRs can be used for optogenetic inhibition of neuronal firing, and potentially for the development of gene therapies to treat neurological and cardiovascular disorders. In this study we identify structural motifs that determine the K+ selectivity of KCRs that provide the foundation for that provide the foundation for elucidating their K+ selectivity mechanism and for their further engineering as optogenetic tools.

Structure of mycobacterial respiratory Complex I

Liang, Y. — 2022-10-05

Oxidative phosphorylation, the combined activity of the electron transport chain (ETC) and adenosine triphosphate synthase, has emerged as a valuable target for the treatment of infection by Mycobacterium tuberculosis and other mycobacteria. The mycobacterial ETC is highly branched with multiple dehydrogenases transferring electrons to a membrane-bound pool of menaquinone and multiple oxidases transferring electrons from the pool. The proton-pumping type I nicotinamide adenine dinucleotide (NADH) dehydrogenase (Complex I) is found at low abundance in the plasma membranes of mycobacteria in typical in vitro culture conditions and is often considered dispensable. We found that growth of Mycobacterium smegmatis in carbon-limited conditions greatly increased the abundance of Complex I and allowed isolation of a rotenone-sensitive preparation of the enzyme. Determination of the structure of the complex by cryoEM revealed the "orphan" two-component response regulator protein MSMEG_2064 as a subunit of the assembly. MSMEG_2064 in the complex occupies a site similar to the proposed redox sensing subunit NDUFA9 in eukaryotic Complex I. An apparent purine nucleoside triphosphate within the NuoG subunit resembles the GTP-derived molybdenum cofactor in homologous formate dehydrogenase enzymes. The membrane region of the complex binds acyl phosphatidylinositol dimannoside, a characteristic three-tailed lipid from the mycobacterial membrane. The structure also shows menaquinone, which is preferentially used over ubiquinone by gram-positive bacteria, in two different positions along the quinone channel and suggests that menaquinone interacts more extensively than ubiquinone with a key catalytic histidine residue in the enzyme.

A CREBZF/Zhangfei isoform activates CHOP during prolonged cellular stress

Li, Y. — 2022-10-11

The basic leucine zipper transcription factor CREBZF (Zhangfei or ZF) was identified through its interaction with Herpes Simplex Virus-1 related cellular protein HCF-1. CREBZF has been implicated in cellular stress responses through its interaction with other proteins, such as CREB3/Luman and ATF4. Here we investigated the production of four CREBZF isoforms, which arise from translational initiation of a downstream AUG at codon 83, and mRNA alternative splicing that adds an IFFFR pentapeptidyl tail to the C-terminus. We found that in addition to transcriptional activation, the short-tailed CREBZF (stZF) isoform was specifically induced by prolonged ER stress treatment. This stZF isoform is a potent transcriptional activator of the pro-apoptotic protein CHOP. Overexpression of stZF activates transcription of CHOP through a CCAAT enhancer binding protein (C/EBP)-ATF site, and promotes apoptosis. We propose that 1) CREBZF is a key component of the Integrated Stress Response (ISR); 2) stZF is essential for the role of CREBZF in inducing CHOP and promoting cell death upon prolonged cellular stress.

Lysosomal swelling and lysis mediate delivery of C3 toxins to their cytoplasmic targets

Grinstein, S. — 2022-10-13

Unlike other cholera-like toxins that contain separate binding/translocation and catalytic subunits, C3-like mono-ADP-ribosyltransferases consist of a single subunit that serves both functions. The manner whereby C3 toxins reach the host cell cytoplasm is poorly understood and was addressed in this study by monitoring the fate of fluorescently-labelled C3larvinA. Following binding to the macrophage membrane in a discontinuous, punctate pattern, the toxin was internalized, traversing the endocytic pathway to reach lysosomes. Strikingly, the lysosomes of C3larvinA-treated cells underwent massive swelling over the course of 1-4 hrs. Lysosomal swelling preceded the extensive rearrangement of the cellular F-actin caused by ADP-ribosylation of cytosolic Rho-GTPases. This suggested that lysosome swelling might be required for escape of the toxin into the cytoplasm where the GTPases reside. Accordingly, preventing swelling by osmotic manipulation or by arresting macropinocytosis precluded the F-actin rearrangement. Toxin-induced swelling was associated with leakage of sulforhodamine B and dextran from the lysosomes, implying membrane rupture or activation of mechano-sensitive pores, enabling the toxin itself to reach the cytosol. Finally, comparison of the cellular traffic and actin remodelling activities of C3larvinA with that of two related toxins, C3larvintrunc and Plx2A, highlighted the importance of the N-terminal 1-helix for lysosomal swelling and successful infection.

Social rank influences relationships between hormones and oxidative stress in a cichlid fish

Culbert, B. M. — 2022-10-17

An individuals social environment can have widespread effects on their physiology, including effects on oxidative stress and hormone levels. Many studies have posited that variation in oxidative stress experienced by individuals of different social ranks might be due to endocrine differences, however, few studies have evaluated this hypothesis. Here, we assessed whether a suite of markers associated with oxidative stress in different tissues (blood, plasma, liver, or gonads) had social rank-specific relationships with circulating testosterone or cortisol levels in males of a cichlid fish, Astatotilapia burtoni. Across all fish, blood DNA damage (a global marker of oxidative stress) and gonadal synthesis of reactive oxygen species (as indicated by NADPH-oxidase (NOX) activity) were lower when testosterone was high. High DNA damage in both the blood and gonads was associated with high cortisol in subordinates, but low cortisol in dominants. Additionally, high cortisol was associated with greater production of reactive oxygen species (greater NOX activity) in both the gonads (dominants only) and liver (dominants and subordinates). In general, high testosterone was associated with lower oxidative stress across both social ranks, whereas high cortisol was associated with lower oxidative stress in dominants and higher oxidative stress in subordinates. Taken together, our results show that differences in the social environment can lead to contrasting relationships between hormones and oxidative stress.

Social memory in female mice is rapidly modulated by 17β-estradiol through ERK and Akt modulation of synapse formation

Sheppard, P. A. S. — 2022-10-25

BackgroundSocial memory is essential to the functioning of a social animal within a group. Estrogens can affect social memory too quickly for classical genomic mechanisms. Previously, 17{beta}-estradiol (E2) rapidly facilitated short-term social memory and increased nascent synapse formation, these synapses being potentiated following neuronal activity. However, what mechanisms underlie and co-ordinate the rapid facilitation of social memory and synaptogenesis are unclear. Here, the necessity of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K) signaling for rapid facilitation of short-term social memory and synaptogenesis was tested. MethodsMice performed a short-term social memory task or were used as task-naive controls. ERK and PI3K pathway inhibitors were infused intra-dorsal hippocampally 5 minutes before E2 infusion. Forty minutes following intrahippocampal E2 or vehicle administration, tissues were collected for quantification of glutamatergic synapse number in the CA1. ResultsDorsal hippocampal E2 rapid facilitation of short-term social memory depended upon ERK and PI3K pathways. E2 increased glutamatergic synapse number (GluA1/bassoon colocalization) in task-performing mice but decreased synapse number in task-naive mice. Critically, ERK signaling was required for synapse formation/elimination in task-performing and task-naive mice, whereas PI3K inhibition blocked synapse formation only in task-performing mice. ConclusionsWhilst ERK and PI3K are both required for E2 facilitation of short-term social memory and synapse formation, only ERK is required for synapse elimination. This demonstrates previously unknown, bidirectional, rapid actions of E2 on brain and behaviour and underscores the importance of estrogen signaling in the brain to social behaviour.

Metals and metal isotopes in insect wings: Implications for diet, geolocation and pollution exposure

Reich, M. S. — 2022-11-03

Anthropogenic activities are exposing insects to abnormal levels of toxic metals, with unknown implications for migratory insects. Simultaneously, metals and metal isotopes have become promising tools for the geolocation of migratory insects. Furthering our understanding of metal cycling in insect tissues is essential, both for the development of metals and metal isotopes as geolocation tools, and for assessing the toxicity risks of metals to insects. We conducted a diet-switching experiment on monarch butterflies (Danaus plexippus) with controlled larval and adult diets to evaluate the dietary and environmental sources of 23 metals and metalloids, strontium isotopes, and lead isotopes to insect wing tissues over a period of 8 weeks. Concentrations of Ca, Co, and Sb differed between the sexes. Ni and Zn bioaccumulated in the insect wing tissues over time, likely from the adult diet, while increases in Al, Cr, Cd, Cu, Fe, and Pb were likely from external sources (i.e., dust aerosols). Bioaccumulation of Pb in the monarch wings was confirmed by Pb isotopes to be from external anthropogenic sources, revealing the potential of Pb isotopes to become an indicator and tracer of metal pollution exposure along migratory paths. Concentrations of Ba, Cs, Mg, Na, Rb, Sr, Ti, Tl, and U appeared to be unaffected by dietary or environmental contamination and should be further developed for geolocation purposes. Strontium isotope ratios remained indicative of the larval diet, at least in males, supporting its potential as a geolocation tool. However, the difference in strontium isotope ratios between sexes, as well as the possibility of external contamination by wetting, requires further investigation. Our results demonstrate the complexity of metal cycling in insects and the need for further investigations, as well as the value of studying metals to develop new tools to quantify pollution exposure, metal toxicity and insect mobility.

Target-distractor competition modulates saccade trajectories in space and object-space

Giuricich, C. — 2022-11-04

Saccade planning and execution can be affected by a multitude of factors present in a target selection task. Recent studies have shown that the similarity between a target and nearby distractors affects the curvature of saccade trajectories, due to target-distractor competition. To further understand the nature of this competition, we varied the distance between and the similarity of complex target and distractor objects in a delayed match-to-sample task to examine their effects on saccade trajectories and better understand the underlying neural circuitry. For trials with short saccadic reaction times (SRTs) when target-distractor competition is still active, we found a robust effect of distance consistent with saccade vector averaging, whereas the effect of similarity suggested the existence of an object-based suppressive surround. At longer SRTs there was sufficient time for competition between the objects to complete and the distractor to be inhibited, which resulted in saccade trajectory deviations exhibiting the effects of a spatial suppressive surround. In terms of similarity, as the target-distractor similarity decreased, the initial saccade angle shifted towards the target, reflecting stronger distractor inhibition. There were no interactions between distance and similarity at any point in the time-course of target-distractor competition. Taken together, saccade trajectories reflect target-distractor competition that is affected independently by both spatial and object-space suppressive surrounds. The differences in saccade trajectories at short and long SRTs distinguish between active and completed decision-making processes. Thus, saccade responses are more beneficial than manual responses in studies of decision-making models. Significance StatementThis is the first study to determine that the distance and similarity between visual objects independently affect saccade trajectories driven by the target-distractor competition process. Thus, spatiotemporal and object identity factors separately feed into saccade planning and execution, resulting in modulations of saccade trajectory metrics which are based on spatial and object-space suppressive surround mechanisms. Furthermore, this modulation of trajectory metrics distinguishes between active and complete decision-making processes. The findings are important for understanding the dynamic networks guiding target selection and are relevant for further development of decision-making models, as well as eye-tracking applications in health and disease.

Alterations in intestinal Proteobacteria and antimicrobial resistance gene burden in individuals administered microbial ecosystem therapeutic (MET-2) for recurrent Clostridioides difficile infection

Rooney, A. M. — 2022-11-04

Intestinal colonisation with pathogens and antimicrobial resistant organisms (AROs) is associated with increased risk of infection. Fecal microbiota transplant (FMT) has successfully been used to cure recurrent Clostridioides difficile infection (rCDI) and to decolonise intestinal AROs. However, FMT has significant practical barriers to implementation. A microbial consortium, microbial ecosystem therapeutic (MET)-2, is an alternative to FMT for the treatment of rCDI. It is unknown whether MET-2 is associated with decreases in pathogens and antimicrobial resistance genes (ARGs). We conducted a post-hoc metagenomic analysis of stool collected from two interventional studies of MET-2 (published) and FMT (unpublished) for rCDI treatment to understand if MET-2 had similar effects to FMT for decreasing pathogens and ARGs as well as increasing anaerobes. Patients were included in the current study if baseline stool had Proteobacteria relative abundance [≥]10% by metagenomic sequencing. We assessed pre- and post-treatment Proteobacteria, obligate anaerobe and butyrate-producer relative abundances and total ARGs. MET-2 and FMT were associated with decreases in Proteobacteria relative abundance as well as increases in obligate anaerobe and butyrate-producer relative abundances. The microbiota response remained stable over 4 or 6 months for MET-2 and FMT, respectively. MET-2, but not FMT, was associated with a decrease in the total number of ARGs. MET-2 is a potential therapeutic strategy for ARO/ARG decolonisation and anaerobe repletion.

Predicting environmental stressor levels with machine learning: a comparison between amplicon sequencing, metagenomics, and total RNA sequencing based on taxonomically assigned data

Hempel, C. A. — 2022-11-18

BackgroundMicrobes are increasingly (re)considered for environmental assessments because they are powerful indicators for the health of ecosystems. The complexity of microbial communities necessitates powerful novel tools to derive conclusions for environmental decision-makers, and machine learning is a promising option in that context. While amplicon sequencing is typically applied to assess microbial communities, metagenomics and total RNA sequencing (herein summarized as omics-based methods) can provide a more holistic picture of microbial biodiversity at sufficient sequencing depths. Despite this advantage, amplicon sequencing and omics-based methods have not yet been compared for taxonomy-based environmental assessments with machine learning. In this study, we applied 16S and ITS-2 sequencing, metagenomics, and total RNA sequencing to samples from a stream mesocosm experiment that investigated the impacts of two aquatic stressors, insecticide and increased fine sediment deposition, on stream biodiversity. We processed the data using similarity clustering and denoising (only applicable to amplicon sequencing) as well as multiple taxonomic levels, data types, feature selection, and machine learning algorithms and evaluated the stressor prediction performance of each generated model for a total of 1,536 evaluated combinations of taxonomic datasets and data-processing methods. ResultsSequencing and data-processing methods had a substantial impact on stressor prediction. While omics-based methods detected much more taxa than amplicon sequencing, 16S sequencing outperformed all other sequencing methods in terms of stressor prediction based on the Matthews Correlation Coefficient. However, even the highest observed performance for 16S sequencing was still only moderate. Omics-based methods performed poorly overall, but this was likely due to insufficient sequencing depth. Data types had no impact on performance while feature selection significantly improved performance for omics-based methods but not for amplicon sequencing. ConclusionAmplicon sequencing might be a better candidate for machine-learning-based environmental stressor prediction than omics-based methods, but the latter require further research at higher sequencing depths to confirm this conclusion. More sampling could improve stressor prediction performance, and while this was not possible in the context of our study, thousands of sampling sites are monitored for routine environmental assessments, providing an ideal framework to further refine the approach for possible implementation in environmental diagnostics.

Dynamics of methane cycling microbiome during methane flux hot moments from riparian buffer systems

Obregon, D. — 2022-11-24

Riparian buffer systems (RBS) are a common agroforestry practice that consists of keeping a forested boundary adjacent to water bodies in agricultural landscapes, thus helping to protect aquatic ecosystems from adverse impacts. Nevertheless, despite the multiple benefits they provide, RBSs can be hotspots of methane emissions since abundant organic carbon and high-water tables are often found in these soils. In southern Ontario, Canada, the rehabilitation of Washington Creeks streambank occurred in 1985. In a recent study, methane (CH4) fluxes were measured biweekly for two years (2017-2018) in four different vegetative riparian areas alongside Washington creek: a rehabilitated tree buffer (RH), a grassed buffer (GRB), an undisturbed deciduous forest (UNF), an undisturbed coniferous forest (CF), and an adjacent agricultural field (AGR) for comparison. Based on methane fluxes in 2018 and hot moments identified, we selected two dates from summer (July 04 and August 15) and use soil sampling from those days to assess the CH4 cycling microbial communities in these RBS. We used qPCR and high-throughput sequencing from both DNA and cDNA to measure the diversity and activity of the methanogen and methanotroph communities. Methanogens were abundant in all riparian soils, including the archaeal genera Methanosaeta, Methanosarcina, Methanomassiliicoccus Methanoreggula, but they were mostly active in UNF soils. Among methanotrophs, Methylocystis was the most abundant taxon in all the riparian sites, except for AGR soils where the methanotrophs community mostly comprised members of rice paddy clusters (RPCs and RPC-1) and upland soil clusters (TUSC and USC). In summary, these results indicate that differences in CH4 fluxes between RBS at Washington creek are influenced by differences in the presence and activity of methanogens, which were higher in the deciduous forest (UNF) soils during hot moments CH4 flux, likely due to high water content in that soils. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/517857v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@1dcde21org.highwire.dtl.DTLVardef@17b59e5org.highwire.dtl.DTLVardef@14adfb0org.highwire.dtl.DTLVardef@a3b411_HPS_FORMAT_FIGEXP M_FIG C_FIG

Antimicrobial susceptibility testing for diverse Malassezia species

Shapiro, R. S. — 2022-12-09

The genus Malassezia is an opportunistic lipid-dependent yeast that is associated with common skin diseases and has recently been associated with Crohns disease and certain cancers. Understanding the susceptibility of Malassezia to diverse antimicrobial agents is crucial for identifying effective antifungal therapies. Here, we tested the efficacy of isavuconazole, itraconazole, terbinafine and artemisinin against three Malassezia species: M. restricta, M. slooffiae, and M. sympodialis, using microbroth dilution techniques. We found antifungal properties for the two previously-unstudied antimicrobials: isavuconazole and artemisinin. Overall all Malassezia species were particularly susceptible to itraconazole, with a MIC range from 0.0015 to 0.1562 M. ImportanceThe Malassezia genus is known to be a cause of skin conditions and has recently been associated with diseases such as Crohns disease, pancreatic ductal carcinoma and breast cancer. This work was completed to assess the susceptibility to a variety of antimicrobial drugs on diverse Malassezia species, in particular Malassezia restricta.

Development and characterization of new patient-derived xenograft (PDX) models of osteosarcoma with distinct metastatic capacities

Schott, C. R. — 2023-01-20

Models to study metastatic disease in rare cancers are needed to advance preclinical therapeutics and to gain insight into disease biology, especially for highly aggressive cancers with a propensity for metastatic spread. Osteosarcoma is a rare cancer with a complex genomic landscape in which outcomes for patients with metastatic disease are poor. As osteosarcoma genomes are highly heterogeneous, a large panel of models is needed to fully elucidate key aspects of disease biology and to recapitulate clinically-relevant phenotypes. We describe the development and characterization of osteosarcoma patient-derived xenografts (PDXs) and a panel of PDX-derived cell lines. Matched patient samples, PDXs, and PDX-derived cell lines were comprehensively evaluated using whole genome sequencing and RNA sequencing. PDXs and PDX-derived cell lines largely maintained the expression profiles of the patient from which they were derived despite the emergence of whole-genome duplication (WGD) in a subset of cell lines. These cell line models were heterogeneous in their metastatic capacity and their tissue tropism as observed in both intravenous and orthotopic models. As proof-of-concept study, we used one of these models to test the preclinical effectiveness of a CDK inhibitor on the growth of metastatic tumors in an orthotopic amputation model. Single-agent dinaciclib was effective at dramatically reducing the metastatic burden in this model. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/524562v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@17ea2a8org.highwire.dtl.DTLVardef@e8fcc7org.highwire.dtl.DTLVardef@2b9f8corg.highwire.dtl.DTLVardef@1f7b802_HPS_FORMAT_FIGEXP M_FIG C_FIG

Novel Pipeline for Large-Scale Comparative Population Genetics

Majoros, S. E. — 2023-01-23

This study determined population genetic structure measures, compared these measures across species with different biological traits; and created efficient, reproducible, reusable programming modules that are publicly available for future research. Cytochrome C Oxidase subunit I gene sequences from Diptera (true fly) species from Greenland and Canada were used as a case study and proof of concept. We hypothesized that population genetic structure measures will be influenced by the biological traits of organisms. Data were pulled from public databases, as well as taxon-specific literature. The R pipeline includes fifteen modules that can be adapted and applied to a diverse set of animal groups, geographic regions, genes, and traits. Habitat, larval diet, geographical distance, latitude, and longitude were all significantly related to population genetic structure in Diptera. Overall, this study has created efficient, reusable bioinformatics modules, as well as provided insight into the factors affecting population genetic structure in Northern fly communities.

The diversity and virulence correlates of metastrongyles in marine mammals

Fischbach, J. R. — 2023-02-04

Metastrongyle lungworms could be particularly detrimental for diving animals such as marine mammals, however little is known of the drivers of pathogenic host-parasite relationships in this group. This systematic review analyzed the diversity of metastrongyles in marine mammals and the host and parasite traits associated with virulence. There have been at least 40 species of metastrongyles described in 66 species of marine mammals. After penalization for study biases, Halocercus hyperoodoni, Otostrongylus circumlitus, Parafilaroides gymnurus, Halocercus brasiliensis, and Stenurus minor were the metastrongyles with the widest host range. Most studies (80.12%, n=133/166) reported that metastrongyles caused bronchopneumonia, while in the cardiovascular system metastrongyles caused vasculitis in nearly half of the studies (45.45%, n=5/11) that assessed these tissues. Metastrongyles were associated with otitis in 23.08% (n=6/26) of the studies. Metastrongyle infection was considered a potential contributory to mortality in 44.78% (n=90/201) of the studies while 10.45% (n=21/201) of these studies considered metastrongyles the main cause of death. Metastrongyle species with a wider host range were more likely to induce pathogenic effects. Metastrongyles can cause significant tissue damage and mortality in marine mammals although virulent host-parasite relationships are dominated by a few metastrongyle species with wider host ranges. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/527020v3_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@9a3e28org.highwire.dtl.DTLVardef@66c8eborg.highwire.dtl.DTLVardef@10cffforg.highwire.dtl.DTLVardef@1c781df_HPS_FORMAT_FIGEXP M_FIG C_FIG

Neural and behavioral correlates of edible cannabis-induced poisoning: characterizing a novel preclinical model

Amissah, R. Q. — 2023-03-15

Accidental exposure to {Delta}9-tetrahydrocannabinol (THC)-containing edible cannabis, leading to cannabis poisoning, is common in children and pets; however, the neural mechanisms underlying these poisonings remain unknown. Therefore, we examined the effects of acute edible cannabis-induced poisoning on neural activity and behavior. Adult Sprague-Dawley rats (6 males, 7 females) were implanted with electrodes in the prefrontal cortex (PFC), dorsal hippocampus (dHipp), cingulate cortex (Cg), and nucleus accumbens (NAc). Cannabis poisoning was then induced by exposure to a mixture of Nutella (6 g/kg) and THC-containing cannabis oil (20 mg/kg). Subsequently, cannabis tetrad and neural oscillations were examined 2, 4, 8, and 24 h after THC exposure. In another cohort (16 males, 15 females), we examined the effects of cannabis poisoning on learning and prepulse inhibition, and the serum and brain THC and 11-hydroxy-THC concentrations. Cannabis poisoning resulted in sex differences in brain and serum THC and 11-hydroxy-THC levels over a 24-h period. It also caused gamma power suppression in the Cg, dHipp, and NAc in a sex- and time-dependent manner. Cannabis poisoning also resulted in hypolocomotion, hypothermia, and anti-nociception in a time-dependent manner and impairments in learning and prepulse inhibition. Our results suggest that the impairments in learning and information processing may be due to the decreased gamma power in the dHipp and PFC. Additionally, most of the changes in neural activity and behavior appear 2 hours after ingestion, suggesting that interventions at or before this time might be effective in reversing or reducing the effects of cannabis poisoning.

Automated high throughput animal DNA metabarcode classification

Porter, T. M. — 2017-11-14

Until now, there has been difficulty assigning names to animal barcode sequences isolated directly from eDNA in a rapid, high-throughput manner, providing a measure of confidence for each assignment. To address this gap, we have compiled nearly 1 million marker gene DNA barcode sequences appropriate for classifying chordates, arthropods, and flag members of other major eukaryote groups. We show that the RDP naive Bayesian classifier can assign the same number of queries 19 times faster than the popular BLAST top hit method and reduce the false positive rate by two-thirds. As reference databases become more representative of current species diversity, confidence in taxonomic assignments should continue to improve. We recommend that investigators can improve the performance of species-level assignments immediately by supplementing existing reference databases with full-length DNA barcode sequences from representatives of local fauna.

Generation of a novel growth-enhanced and reduced environmental impact transgenic pig strain

Zhang, X. — 2018-01-20

In pig production, insufficient feed digestion causes excessive nutrients such as phosphorus and nitrogen, which are then released to the environment. To address the issue of environmental emissions, we have established transgenic pigs harboring a single-copy quad-cistronic transgene and simultaneously expressing three microbial enzymes, {beta}-glucanase, xylanase, and phytase in the salivary glands. All the transgenic enzymes were successfully expressed, and the digestion of non-starch polysaccharides (NSPs) and phytate in the feedstuff was enhanced. Fecal nitrogen and phosphate outputs were reduced by 23%-46%, and growth rate improved by 23.4% (gilts) and 24.4% (boars) when the pigs were fed on a corn and soybean-based diet and high-NSP diet. The transgenic pigs showed a 11.5%- 14.5% improvement in feed conversion rate compared to the age-matched wild-type littermates. These findings indicate that transgenic pigs are promising resources for improving feed efficiency and reducing nutrient emissions to the environment.

Convergence of socio-ecological dynamics in disparate ecological systems under strong coupling to human social systems

Sigdel, R. — 2018-04-06

It is widely recognized that coupled socio-ecological dynamics can be qualitatively different from the dynamics of social or ecological systems in isolation from one another. The influence of the type of ecological dynamics on the dynamics of the larger socio-ecological system is less well studied, however. Here, we carry out such a comparison using a mathematical model of a common pool resource problem. A population must make decisions about harvesting a renewable resource. Individuals may either be cooperators, who harvest at a sustainable level, or defectors, who over-harvest. Cooperators punish defectors through social ostracism. Individuals can switch strategies according the costs and benefits of harvesting and the strength of social ostracism. These mechanisms are represented by a differential equation for social dynamics which is coupled to three different types of resource dynamics: logistic growth, constant inflow, and threshold growth. We find that when human influence is sufficiently weak, the form of natural dynamics leaves a strong imprint on the socio-ecological dynamics, and human social dynamics are qualitatively very different from natural dynamics. However, stronger human influence introduces a broad intermediate parameter regime where dynamical patterns converge to a common type: the three types of ecological systems exhibit similar dynamics, but also, social and ecological dynamics strongly mirror one another. This is a consequence of stronger coupling and is reminiscent of synchrony from other fields, such as the classic problem of coupled oscillators in physics. Socio-ecological convergence has implications for how we understand and manage complex socio-ecological systems. In an era of growing human influence on ecological systems, further empirical and theoretical work is required to determine whether socio-ecological convergence is present in real systems.

Trend and pattern of antimicrobial resistance in molluscan Vibrio species sourced to Canadian estuaries

Banerjee, S. K. — 2018-04-21

Emergence of antimicrobial resistance (AMR) in foodborne bacteria is a growing concern worldwide. AMR surveillance is a key element in understanding the implications resulting from the use of antibiotics for therapeutic as well as prophylactic needs. The emergence and spread of AMR in foodborne human pathogens is an indirect health hazard. This surveillance study reports the trend and pattern of AMR detected in Vibrio species isolated from molluscs harvested in Canada, between 2006 and 2012, against 19 commonly used antibiotics. Five common antibiotics, ampicillin, cephalothin, erythromycin, kanamycin and streptomycin, predominantly contributed to AMR including multi-drug resistance (MDR) in the molluscan Vibrio spp. isolated in 2006. A prospective follow-up analysis of these drugs showed a declining trend in the frequency of MDR/AMR-Vibrio spp. in subsequent years until 2012. The observed decline appears to have been influenced by the specific downturn in resistance to the aminoglycosides, kanamycin and streptomycin. Frequently observed MDR/AMR-Vibrio spp. in seafood is a potential health concern associated with seafood consumption. Our surveillance study provided an indication of the antibiotics that challenged the marine bacteria, sourced to Canadian estuaries, during and/or prior to the study period.

Anti-diuretic activity of a CAPA neuropeptide can compromise Drosophila chill tolerance

MacMillan, H. A. — 2018-05-31

For insects, chilling injuries that occur in the absence of freezing are often related to a systemic loss of ion and water balance that leads to extracellular hyperkalemia, cell depolarization, and the triggering of apoptotic signalling cascades. The ability of insect ionoregulatory organs (e.g. the Malpighian tubules) to maintain ion balance in the cold has been linked to improved chill tolerance, and many neuroendocrine factors are known to influence ion transport rates of these organs. Injection of micromolar doses of CAPA (an insect neuropeptide) have been previously demonstrated to improve Drosophila cold tolerance, but the mechanisms through which it impacts chill tolerance are unclear, and low doses of CAPA have been demonstrated to cause anti-diuresis in other insects, including dipterans. Here, we provide evidence that low (fM) and high ({micro}M) doses of CAPA impair and improve chill tolerance, respectively, via two different effects on Malpighian tubule ion and water transport. While low doses of CAPA are anti-diuretic, reduce tubule K+ clearance rates and reduce chill tolerance, high doses facilitate K+ clearance from the haemolymph and increase chill tolerance. By quantifying CAPA peptide levels in the central nervous system, we estimated the maximum achievable hormonal titres of CAPA, and found evidence to suggest that CAPA may function as an anti-diuretic peptide in Drosophila. We provide the first evidence of a neuropeptide that can negatively affect cold tolerance in an insect, and the first evidence of CAPA as an anti-diuretic peptide in this ubiquitous insect model.nnSummary StatementMany insects ion balance in the cold. We show how one neuropeptide can slow ion transport and reduce the cold tolerance of a fly.

Can Drosophila melanogaster tell who’s who?

Schneider, J. — 2018-06-08

Drosophila melanogaster are known to live in a social but cryptic world of touch and odours, but the extent to which they can perceive and integrate visual information is a hotly debated topic. Some researchers fixate on the limited resolution of D. melanogasters optics, others on their seemingly identical appearance; yet there is evidence of individual recognition and surprising visual learning in flies. Here, we apply machine learning and show that individual D. melanogaster are visually distinct. We also use the striking similarity of Drosophilas visual system to current convolutional neural networks to theoretically investigate D. melanogasters capacity for visual understanding. We find that, despite their limited optical resolution, D. melanogasters neuronal architecture has the capability to extract and encode a rich feature set that allows flies to re-identify individual conspecifics with surprising accuracy. These experiments provide a proof of principle that Drosophila inhabit in a much more complex visual world than previously appreciated.nnAuthor summaryIn this paper, we determine a proof of principle for inter-individual recognition in two parts; is there enough information contained in low resolution pictures for inter-fly discrimination, and if so does Drosophilas visual system have enough capacity to use it. We show that the information contained in a 29x29 pixel image (number of ommatidia in a fly eye) is sufficient to achieve 94% accuracy in fly re-identification. Further, we show that the fly eye has the theoretical capacity to identify another fly with about 75% accuracy. Although it is unlikely that flies use the exact algorithm we tested, our results show that, in principle, flies may be using visual perception in ways that are not usually appreciated.

Aptamer-based biosensor for food allergen determination using graphene oxide/gold nanocomposite on a paper-assisted analytical device

Tah, A. — 2018-07-09

The detection of allergens in food are currently conducted by techniques that are time-consuming and complicated which can deter consistent sampling for allergens, which could potentially cause an anaphylactic shock in the consumer by cross-contamination. The need for a technique that is rapid, on-site, cost-effective, disposable, highly sensitive and accurate to identify these molecules urges the development of a point-of-care device. The aim of this work is to develop a microfluidic paper-assisted analytical device (PAD) using hydrophobic channels, set by a wax printer on filter paper, and functionalized gold nanoparticles (AuNP) to help identify the allergens arachin (Ara h 1) for peanuts, {beta}-lactoglobulin ({beta} LG) for milk, and tropomyosin (Pen a 1) for shrimp and other shellfish presence by a colorimetric test. Synthesized AuNP were conjugated with biotinylated aptamers, using the biotin-streptavidin interaction, to make the specific detection of target allergens. Functionalized AuNP are incubated with the sample and are absorbed by graphene oxide (GO), creating GO-AuNP complexes, if the aptamers have not become structured due to conjugation with allergenic proteins. The PAD device is used to filter the resultant mixture which provides superior sensitivity to detect the allergens present down to the nanogram range (allergens were measured from 25 nM - 1000 nM with a LOD of 7.8 nM, 12.4 nM and 6.2 nM for peanut, milk and shrimp allergens respectively), in contrast to the microgram range of commonly used enzymatic immunoassays. The simple color indicator, varying from clear to pink in the presences of allergens allows the readout to be utilized without the need for highly specific equipment or training. Alternatively, the results can be quantified by taking a picture and measuring the color. This presented PAD can provide results in real time and has the potential to become a rapid, low-cost, and accurate portable point-of-care device to avoid cross-reactivity of food-borne allergens.

Over 2.5 million COI sequences in GenBank and growing

Porter, T. M. — 2018-06-22

The increasing popularity of cytochrome c oxidase subunit 1 (COI) DNA metabarcoding warrants a careful look at the underlying reference databases used to make high-throughput taxonomic assignments. The objectives of this study are to document trends and assess the future usability of COI records for metabarcode identification. Over 2.5 million COI sequences were found in GenBank, half of which were fully identified to the species rank. From 2003 to 2017, the number of COI Eukaryote records deposited has grown by two orders of magnitude representing a nearly 42-fold increase in unique species. For fully identified records, 92% are at least 500 bp in length, 74% have a country annotation, and 51% have latitude-longitude annotations. To ensure the future usability of COI records in GenBank we suggest: 1) Improving the geographic representation of COI records 2) Improving the cross-referencing of COI records in the Barcode of Life Data System and GenBank to facilitate consolidation and incorporation into existing bioinformatic pipelines, 3) Adherence to the minimum information about a marker gene sequence guidelines, and 4) Integrating metabarcodes from eDNA and mixed community studies with existing sequences. COI metabarcoders are normally considered consumers of taxonomic data. Here we discuss the potential for taxonomists to reverse this pattern and instead mine metabarcode data to guide species discovery. The growth of COI reference records over the past 15 years has been substantial and is likely to be a resource across many fields for years to come.

mTORC1 activity is dispensable for synthesis of KSHV lytic proteins

Pringle, E. S. — 2018-06-26

Herpesvirus genomes are decoded by host RNA polymerase II, generating messenger ribonucleic acids (mRNAs) that are post-transcriptionally modified and exported to the cytoplasm. These viral mRNAs have 5'-m7GTP caps and poly-adenylate (polyA) tails that should permit assembly of canonical eukaryotic initiation factor 4F (eIF4F) cap-binding complexes to initiate protein synthesis. However, we have shown that chemical disruption of eIF4F does not impede KSHV lytic replication, suggesting that alternative translation initiation factors (TIFs) support viral protein synthesis. Here we show that eIF4F and an alternative TIF known as eIF3d both contribute to the accumulation of viral proteins during KSHV lytic replication, with eIF3d supporting accumulation of viral proteins when eIF4F is depleted and vice-versa. However, eIF3d depletion greatly diminished release of infectious virions even when eIF4F was available to compensate for lost translation initiation capacity. This viral replication failure in the absence of eIF3d correlated with diminished accumulation of many enzymes in the cholesterol biosynthetic pathway and reduced accumulation of intracellular cholesterol. Moreover, we demonstrated that eIF3d was required to support efficient translation of essential cholesterol biosynthesis enzymes SQLE and MVK during lytic replication. These findings position eIF3d as a critical TIF not only required to support efficient translation of KSHV lytic mRNAs during times of need, but also to support the accumulation of intracellular cholesterol required to successfully complete an infectious cycle.

Using eDNA to biomonitor the fish community in a tropical oligotrophic lake

Valdez-Moreno, M. — 2018-07-23

Environmental DNA (eDNA) is an effective approach for detecting vertebrates and plants, especially in aquatic ecosystems, but prior studies have largely examined eDNA in cool temperate settings. By contrast, this study employs eDNA to survey the fish fauna in tropical Lake Bacalar (Mexico) with the additional goal of assessing the possible presence of invasive fishes, such as Amazon sailfin catfish. Sediment and water samples were collected from eight stations in Lake Bacalar on three occasions over a 4-month interval. Each sample was stored in the presence or absence of lysis buffer to compare eDNA recovery. Short fragments (184-187 bp) of the cytochrome c oxidase I (COI) gene were amplified using fusion primers and then sequenced on Ion Torrent PGM and S5 before their source species were determined using a custom reference sequence database constructed on BOLD. In total, eDNA sequences were recovered from 75 species of vertebrates including 47 fishes, 15 birds, 7 mammals, 5 reptiles, and 1 amphibian. Although all species are known from this region, 6 fish species represent new records for the study area, while 2 require verification. Sequences for five species (2 birds, 2 mammals, 1 reptile) were only detected from sediments, while sequences from 52 species were only recovered from water. Because DNA from the Amazon sailfin catfish was not detected, we used a mock eDNA experiment to confirm our methods were appropriate for its detection. We developed protocols that enabled the recovery of eDNA from tropical oligotrophic aquatic ecosystems, and confirmed their effectiveness in detecting diverse species of vertebrates including an invasive species of Amazon catfish.

Risks of exposure to systemic insecticides in agricultural soil in Ontario, Canada for the hoary squash bee (Peponapis pruinosa) and other ground-nesting bee species

Chan, D. S. W. — 2018-10-31

Insecticide exposure is an environmental factor of concern relating to pollinator health. Recent increases in use of systemic insecticides, particularly neonicotinoids, have led to considerable research into the potential impacts for bees of exposure to these insecticide residues via the nectar and pollen of treated crops or contaminated wild flowers. However, although the majority of bee species are ground-nesting, the risk of exposure to insecticides in soil has not yet been evaluated. Here we use the hoary squash bee (Peponapis pruinosa) as a model system to provide the first evaluation of the risk of exposure to insecticide residues in soil to ground-nesting solitary bee species. The evaluation assessed agrochemical residues from the nectar, pollen and soil from Cucurbita-crops (e.g. squash and pumpkin) grown in southern Ontario in 2016, and soil insecticide residue data collected from field crops by the Ontario government. Systemic insecticide residues were rarely detected in the nectar and pollen of Cucurbita-crops in 2016, and hazard assessment deemed these potential routes of exposure to be non-hazardous for honey bee lethal dose endpoints. In contrast, quantifiable pesticide residues were frequently detected in soil suggesting this route of exposure to be hazardous for honey bee lethal dose endpoints, leading to further assessment using probabilistic risk assessments. Concentrations of clothianidin, imidacloprid, thiamethoxam (neonicotinoid insecticides) and chlorantraniliprole (an anthranilic diamide insecticide) in soil samples were plotted to produce an environmental exposure distribution for each insecticide. A honey bee LC50 and a solitary bee LC50 endpoint were converted to exposure endpoints using the amount of soil excavated by hoary squash bees during nest construction, for both acute and chronic exposure scenarios. The probability of exceedance of each exposure endpoint was calculated and compared to an acceptable risk threshold (i.e. 5% exceedance). In the acute exposure scenario, risk to hoary squash bees was below acceptable threshold levels for all residues evaluated using the honey bee LC50, but exceeded the threshold for clothianidin and imidacloprid using the solitary bee LC50. In the chronic exposure scenario, risk from exposure to clothianidin and imidacloprid exceeded the threshold for both the honey bee and solitary bee LC50s, and for chlorantraniliprole risk only exceeded the threshold for the solitary bee LC50. Using the hoary squash bee as a model, the wider implications for other ground-nesting bees that pollinate crops was explored using insecticide residues from soil samples taken from Ontario field crops. Probabilistic risk assessments suggest that risk to ground-nesting bees is high from clothianidin residues in soils, even when exposure is acute, and substantial for both thiamethoxam and imidacloprid under chronic exposure scenarios. These results demonstrate the urgent need to consider direct exposure to pesticides in soil for ground-nesting bees as part of risk assessments and provides a potential model for evaluating risk from this type of exposure.

Conditioned place avoidance using encapsulated calcium propionate as an appetite suppressant for broiler breeders

Arrazola, A. — 2018-10-11

Broiler breeders, the parent stock of meat chicks, are feed-restricte throughout rearing to avoid obesity-related problems in their health and reproductive performance. Broiler breeders often show signs of chronic hunger, lack of satiety and feeding frustration, and the development of alternative feeding strategies has investigated the inclusion of calcium propionate (CaP) as an appetite suppressant. However, the mechanisms involved in the reduction of voluntary feed intake are unknown, but are thought to be due to low palatability, gastrointestinal discomfort, or both. The objective of this experiment was to examine the effect of CaP as an appetite suppressant on the experience of a negative affective state, using a conditioned place preference test. Twenty four broiler breeders were trained to associate the consumption of CaP or a placebo pill with a red or blue place, depending on inherent colour preference. Pullets consumed two pills followed by 20 g feed allotment. The CaP pill contained 160 mg of CaP and the placebo pill had 160 mg of feed. Conditioning lasted for 90 min/pullet/day over 8 consecutive days at 7 and 9 weeks of age, and pullets choice was tested in a T-maze twice on two consecutive days at both 8 and 10 weeks of age. Data were analysed using a linear mixed regression model, with pen nested in the model and age as a repeated measure. Pullets were less likely to choose the place conditioned with the consumption of CaP (P<0.05) and the preference of the placebo linearly increased with training sessions (P<0.05). These results suggest that calcium propionate as an appetite suppressant can induce a negative affective state, with the lower feed intake resulting from a conditioned response to the negative effect of calcium propionate rather than to satiety.

Is physiological stress state reflected in acoustic structure of vocalizations? An experimental test in wild North American red squirrels

Sehrsweeney, M. — 2018-10-31

Acoustic signaling is an important means by which animals communicate both stable and labile characteristics. Although it is widely appreciated that vocalizations can convey information on labile state, such as fear and aggression, very few studies have experimentally examined the acoustic expression of short-term stress state. The transmission of such information about physiological state could have broad implications, potentially allowing other individuals to modify their behavior or life history traits in response to this public information. North American red squirrels (Tamiasciurus hudsonicus) produce vocalizations known as rattles that advertise territorial ownership. We examined the influence of changes in physiological stress state on rattle acoustic structure through the application of a stressor (trapping and handling the squirrels) and by provisioning squirrels with exogenous glucocorticoids (GCs). We characterized the acoustic structure of rattles emitted by these squirrels by measuring rattle duration, mean frequency, and entropy. Our results provide mixed evidence that rattles show a "stress signature". When squirrels were trapped and handled, they produced rattles that were longer in duration with a higher frequency and increased entropy. However, squirrels that were administered exogenous GCs had similar rattle duration, frequency, and entropy as squirrels that received control treatments and unmanipulated (unfed) squirrels. Our results indicate that short-term stress does affect the acoustic structure of vocalizations, but elevated circulating GC levels are not solely responsible for such changes.

Indirect genetic effects clarify how traits can evolve even when fitness does not

Fisher, D. N. — 2018-10-31

There are many situations in nature where we expect traits to evolve but not necessarily for mean fitness to increase. However, these scenarios are hard to reconcile simultaneously with Fishers Fundamental Theorem of Natural Selection and the Price identity. The consideration of indirect genetic effects on fitness reconciles these fundamental theorems with the observation that traits sometimes evolve without any adaptation, by explicitly considering the correlated evolution of the social environment, which is a form of transmission bias. While transmission bias in the Price identity is often assumed to be absent, here we show that explicitly considering indirect genetic effects as a form of transmission bias for fitness has several benefits: 1) it makes clear how traits can evolve while mean fitness remains stationary, 2) it reconciles the fundamental theorem of natural selection with the evolution of maladaptation, 3) it explicitly includes density-dependent fitness through negative social effects that depend on the number of interacting conspecifics, and 4) its allows mean fitness to evolve even when direct genetic variance in fitness is zero, if related individuals interact and/or if there is multilevel selection. In summary, considering fitness in the context of indirect genetic effects aligns important theorems of natural selection with many situations observed in nature and provides a useful lens through which we might better understand evolution and adaptation.

Metabolism-Induced Oxidative Stress and DNA Damage Selectively Trigger Genome Instability in Polyploid Cells

Thomson, G. J. — 2018-11-29

Understanding the forces impacting genome stability is important for diverse processes such as tumorigenesis and reproductive biology. The pathogenic yeast Candida albicans displays unusual genome dynamics in which tetraploid cells, but not diploid cells, become unstable when grown on a glucose-rich pre-sporulation medium. Here, we reveal that C. albicans polyploid cells are metabolically hyperactive on this medium as evidenced by increased expression of metabolic genes as well as higher rates of fermentation and oxidative respiration. These cells also show elevated levels of reactive oxygen species (ROS), activate the ROS-responsive transcription factor Cap1, and accrue DNA double-strand breaks. Suppression of ROS levels reduced oxidative stress, DNA damage and chromosome instability. These studies reveal how metabolic flux can generate endogenous ROS, triggering DNA damage and genome instability in polyploid, but not diploid, cells. We discuss parallels with metabolism-induced instability in cancer cells and propose that ROS-induced DNA damage could have facilitated ploidy cycling in eukaryotes prior to the evolution of meiosis.

Comparing the Rat Grimace Scale and a composite behaviour score in rats

Klune, C. — 2018-12-07

There is a growing interest in the use of voluntarily displayed ongoing behaviours in laboratory animals to assess the pain experience. In rats, two behavioural pain scales, the Rat Grimace Scale (RGS, a facial expression scale) and a composite behaviour score (CBS, a behavioural ethogram reliant on postural changes), are both promising pain assessment methods. Both scales have been used to assess pain in a laparotomy model, however, they have never been compared directly and the knowledge of how different analgesics may affect these two scales is limited. This study aimed to provide a comparison to discriminate the temporal and analgesic response in a laparotomy model. Female Wistar (n = 26) and Sprague Dawley rats (n = 26) were block randomized to receive saline, meloxicam (2 mg/kg) or buprenorphine (0.05 mg/kg) 30 minutes before a laparotomy model. Rats were video-recorded before surgery (BL) and at 30, 150, 270, and 390 minutes post-operatively. Videos were assessed according to both scales by a trained, blinded observer. Both CBS and RGS scores increased significantly at all post surgical timepoints in the saline group. Post-surgical CBS scores did not increase significantly above baseline levels in the groups given meloxicam or buprenorphine. However, the RGS scores only remained low in the buprenorphine group while scores increased significantly in the meloxicam group, to a similar degree as in the saline group. These findings suggest that the CBS is more sensitive to the analgesic effects of NSAIDs than the RGS.

Multiple species drive flexible lake food webs with warming

Bartley, T. J. — 2018-12-17

Climate change is rewiring the food webs that determine the fate of diverse ecosystems. Mobile generalist consumers are responding to climate change by rapidly shifting their behaviour and foraging, driving food webs to flex. Although these responsive generalists form a key stabilizing module in food web structure, the extent to which they are present throughout whole food webs is largely unknown. Here, we show that multiple species comprising key trophic roles drive flexible lake food webs with warming. By examining lakes that span a 7{degrees}C air temperature gradient, we found significant reductions in nearshore derived carbon and nearshore habitat use with increased temperature in three of four fish species. We also found evidence that the response of lake trout to increased temperatures may reduce their biomass and cascade to release their preferred prey, the pelagic forage fish cisco. Our results suggest that climate warming will shift lake food webs toward increased reliance on offshore habitats and resources. We argue that species across trophic levels broadly couple lake macrohabitats, suggesting that potentially stabilizing responsive consumers are present throughout food webs. However, climate change appears to limit their ability to responsively forage, critically undermining a repeated stabilizing mechanism in food webs.

Drivers of human gut microbial community assembly: Coadaptation, determinism and stochasticity

Oliphant, K. — 2018-12-19

Microbial community assembly is a complex process shaped by multiple factors, including habitat filtering, species assortment and stochasticity. Understanding the relative importance of these drivers would enable scientists to design strategies initiating a desired reassembly for e.g., remediating low diversity ecosystems. Here, we aimed to examine if a human fecal-derived defined microbial community cultured in bioreactors assembled deterministically or stochastically, by completing replicate experiments under two growth medium conditions characteristic of either high fiber or high protein diets. Then, we recreated this defined microbial community by matching different strains of the same species sourced from distinct human donors, in order to elucidate whether coadaptation of strains within a host influenced community dynamics. Each defined microbial ecosystem was evaluated for composition using marker gene sequencing, and for behaviour using 1H-NMR based metabonomics. We found that stochasticity had the largest influence on the species structure when substrate concentrations varied, whereas habitat filtering greatly impacted the metabonomic output. Evidence of coadaptation was elucidated from comparisons of the two communities; we found that the artificial community tended to exclude saccharolytic Firmicutes species and was enriched for metabolic intermediates, such as Stickland fermentation products, suggesting overall that polysaccharide utilization by Firmicutes is dependent on cooperation.

Seed masting causes fluctuations in optimum litter size and lag load in a seed predator

McAdam, A. — 2018-12-20

The episodic production of large seed crops by some perennial plants, is referred to as masting and is known to increase seed escape by alternately starving and swamping seed predators. These pulses of resources, however, might also act as an agent of selection on the life histories of seed predators, which could indirectly enhance seed escape by inducing an evolutionary load on seed predator populations. Lag loads in seed predators could result from mast-induced shifts in optimum phenotypes that exceed the capacity of seed predators to adaptively track optimum phenotypes through phenotypic plasticity. Alternatively, masting could generate mismatches in selection across generations, where adaptation to the parental environment leads to maladaptation in the offspring environment. Here we measured natural selection on female North American red squirrels (Tamiasciurus hudsonicus) across 28 years and five white spruce (Picea glauca) masting events. Red squirrel litter sizes were similar to optimum litter sizes during non-mast years, but were well below optimum litter sizes during resource-rich mast years. Mast events, therefore caused selection for larger litters ({beta} = 0.25) and a lag load (L = 0.25) on red squirrels during mast years. Furthermore, we found that the annual fitness of spruce trees was negatively related to the local density of squirrels during mast years, indicating that the observed lag load on squirrels enhanced the number of spruce cones escaping squirrel predation. Although, the frequency of mast events and the demography of red squirrels were such that offspring and parents often experienced opposite environments with respect to the mast, we found no effect of environmental mismatches across generations on either offspring survival or population growth. Instead, squirrels plastically increased litter sizes in anticipation of mast events, which partially, although not completely, reduced the lag load resulting from this change in food availability. Variable selection on litter size caused by white spruce mast events, therefore, induced a lag load on the population of red squirrels that was not affected by whether individual squirrels were born during mast (matching) or non-mast (mismatching) conditions.

Soybean Haplotype Map (GmHapMap): A Universal Resource for Soybean Translational and Functional Genomics

Torkamaneh, D. — 2019-01-29

Here we describe the first worldwide haplotype map for soybean (GmHapMap) constructed using whole-genome sequence data for 1,007 Glycine max accessions and yielding 15 million variants. The number of unique haplotypes plateaued within this collection (4.3 million tag SNPs) suggesting extensive coverage of diversity within the cultivated germplasm. We imputed GmHapMap variants onto 21,618 previously genotyped (50K array/210K GBS) accessions with up to 96% success for common alleles. A GWAS performed with imputed data enabled us to identify a causal SNP residing in the NPC1 gene and to demonstrate its role in controlling seed oil content. We identified 405,101 haplotypes for the 55,589 genes and show that such haplotypes can help define alleles. Finally, we predicted 18,031 putative loss-of-function (LOF) mutations in 10,662 genes and illustrate how such a resource can be used to explore gene function. The GmHapMap provides a unique worldwide resource for soybean genomics and breeding.

Evaluating the within-host dynamics of Ranavirus infection with mechanistic disease models and experimental data

Mihaljevic, J. R. — 2019-02-28

Mechanistic models are critical for our understanding of both within-host dynamics (i.e., pathogen population growth and immune system processes) and among-host dynamics (i.e., transmission). Rarely, however, have within-host models been synthesized with data to infer processes, validate hypotheses, or generate new theories. In this study we use mechanistic models and empirical, time-series data of viral titer to better understand the growth of ranaviruses within their amphibian hosts and the immune dynamics that limit viral replication. Specifically, we fit a suite of potential models to our data, where each model represents a hypothesis about the interactions between viral growth and immune defense. Through formal model comparison, we find a parsimonious model that captures key features of our time-series data: the viral titer rises and falls through time, likely due to an immune system response, and that the initial viral dosage affects both the peak viral titer and the timing of the peak. Importantly, our model makes several predictions, including the existence of long-term viral infections, that can be validated in future studies.

Thermal preferences drive behavioural responses but not biomass responses of predatory fishes to lake morphometry

Bartley, T. J. — 2019-03-09

Top predators responses to environmental conditions shape food web architecture and influence ecosystem structure and stability. Yet the impacts of fundamental properties like ecosystem size and morphometry on top predators behaviour are poorly understood. We examined how lake morphometry impacts the behaviour (inferred by depth use) of three key fish top predators--the cold-adapted lake trout, the cool-adapted walleye, and the warm-adapted smallmouth bass-- which can each strongly impact local food web structure. We used catch-per-unit-effort data from nearly 500 boreal lakes of Ontario, Canada to evaluate the role of thermal preference in dictating mean depth of capture and biomass index in response to lake morphometry. We found evidence that thermal preferences influence how species depth use and biomass changed with lake size, proportion of littoral area, and maximum lake depth, although we found no relationship with lake shape. However, found no strong evidence that lake morphology influences these species biomasses, despite theory that predicts such a relationship. Our results suggest that some aspects of lake morphometry can alter habitat accessibility and productivity in ways that influence the behaviour and biomass of these top predator species depending on their thermal preferences. Our results have implications for how lake food webs expand and contract with lake morphometry and other key abiotic factors. We argue that several key abiotic factors likely drive top predator depth use in ways that may shape local food web structure and play an important role in determining the ultimate fate of ecosystems with environmental change.

Territory acquisition mediates the influence of predators and climate on juvenile red squirrel survival

Hendrix, J. G. — 2019-04-02

1) Juvenile survival to first breeding is a key life history stage. Survival through this period can be particularly challenging when it coincides with harsh environmental conditions like winter climate or food scarcity, and so cohort survival can be highly variable. However, the small size and dispersive nature of juveniles makes studying their survival difficult.n2) In territorial species, a key life history event is the acquisition of a territory. A territory is expected to enhance survival, but how it does so, possibly through mediating mortality, is not often identified. We tested how the timing of territory acquisition influenced the survival of juvenile North American red squirrels Tamiasciurus hudsonicus, hereafter red squirrels, and how the timing of this event mediated sources of mortality. We hypothesized that securing a territory prior to the caching season would reduce juvenile susceptibility to predation or climatic factors over winter.n3) Using 27 years of data on the survival of individually-marked juvenile red squirrels, we tested how the timing of territory acquisition influenced survival, whether the population density of red squirrel predators and mean temperature over winter were related to individual survival probability, and if territory ownership mediated these effects.n4) Juvenile survival was lower in years of high predator abundance and in colder winters. Autumn territory owners were less susceptible to lynx Lynx canadensis, and possibly mustelid Mustela and Martes spp., predation. Autumn territory owners had lower survival in colder winters, while non-owners had higher survival in cold winters.n5) Our results show how the timing of a life history event like territory acquisition can directly affect survival and also mediate the effects of biotic and abiotic factors later in life. This engenders a better understanding of the fitness consequences of the timing of key life history events.

Individual variation in phenotypic plasticity of the stress axis

Guindre-Parker, S. — 2019-04-07

Phenotypic plasticity--one individuals capacity for phenotypic variation under different environments--is critical for organisms facing fluctuating conditions within their lifetime. North American red squirrels (Tamiasciurus hudsonicus) experience drastic among-year fluctuations in conspecific density. This shapes juvenile competition over vacant territories and overwinter survival. To help young cope with competition at high densities, mothers can increase offspring growth rates via a glucocorticoid-mediated maternal effect. However, this effect is only adaptive under high densities, and faster growth often comes at a cost to longevity. While experiments have demonstrated that red squirrels can adjust hormones in response to fluctuating density, the degree to which mothers differ in their ability to regulate glucocorticoids across changing densities remains unknown--little is known about within-individual plasticity in endocrine traits relative to among-individual variation. Findings from our reaction norm approach revealed significant individual variation not only in a female red squirrels mean endocrine phenotype, but also in endocrine plasticity in response to changes in local density. Future work on the proximate and ultimate drivers of variation in the plasticity of endocrine traits and maternal effects is needed, particularly in free-living animals experiencing fluctuating environments.

Coherent whole food web responses to outbreaking spruce budworm

Greyson-Gaito, C. J. — 2019-04-26

The world is astoundingly variable, and individuals to whole communities must respond to variability to survive. One example of natures variability is the massive fluctuations in spruce budworm (Choristoneura fumiferana Clemens, Lepidoptera: Tortricidae) populations that occur over 35 years. We examined how the parasitoid community altered its parasitism of budworm and other caterpillar species in response to these fluctuations. Budworm and other caterpillar species were sampled from balsam fir in three plots for 14 years in Atlantic Canada, and then reared to identify any emerging parasitoids. We found that the parasitoid community showed a simple linear, indiscriminate response (i.e., no preference, where densities purely dictated parasitism rates) to changes in budworm densities relative to other caterpillar species on balsam fir. We also observed strong changes in topology and distributions of interaction strengths between the parasitoids, budworm and other caterpillar species as budworm densities fluctuated. Our study contributes to the suggestion that hardwood trees are a critical part of the budworm-parasitoid food web, where parasitoids attack other caterpillar species on hardwood trees when budworm populations are low. Taken together, our study shows that a parasitoid community collectively alters species interactions in response to variable budworm densities, fundamentally shifting food web pathways.

DEAD-box protein family member DDX28 is a negative regulator of HIF-2α and eIF4E2-directed hypoxic translation

Evagelou, S. L. — 2019-05-09

Hypoxia occurs when there is a deficiency in oxygen delivery to tissues and is connected to physiological and pathophysiological processes such as embryonic development, wound healing, heart disease and cancer. The master regulators of oxygen homeostasis in mammalian cells are the heterodimeric hypoxia-inducible transcription factors HIF-1 and HIF-2. The oxygen-labile HIF-2 subunit has not only been implicated in transcription, but also as a regulator of eIF4E2-directed hypoxic translation. Here, we have identified the DEAD-box protein family member DDX28 as a novel interactor and negative regulator of HIF-2 that suppresses its ability to activate eIF4E2-directed translation. We demonstrate that stable silencing of DDX28 via shRNA in hypoxic human U87MG glioblastoma cells caused an increase, relative to control, to: HIF-2 protein levels, the ability of eIF4E2 to bind the m7GTP cap structure, and the translation of select eIF4E2 target mRNAs. DDX28 depletion elevated both nuclear and cytoplasmic HIF-2, but HIF-2 transcriptional activity did not increase possibly due to its already high nuclear abundance in hypoxic control cells. Depletion of DDX28 conferred a proliferative advantage to hypoxic, but not normoxic cells, which is likely a consequence of the translational upregulation of a subset of hypoxia-response mRNAs. DDX28 protein levels are reduced in several cancers, including glioma, relative to normal tissue. Therefore, we uncover a regulatory mechanism for this potential tumor suppressor in the repression of HIF-2- and eIF4E2-mediated translation activation of oncogenic mRNAs.

Stirred Suspension Bioreactor Culture of Porcine Induced Pluripotent Stem Cells

Burrell, K. — 2019-05-20

Induced pluripotent stem cells (iPSCs) are an attractive cell source for regenerative medicine and the development of therapies, as they can proliferate indefinitely under defined conditions and differentiate into any cell type in the body. Large scale expansion of cells is limited in adherent culture, making it difficult to obtain adequate cell numbers for research. It has been previously shown that stirred suspension bioreactors (SSBs) can be used to culture mouse and human stem cells. Pigs are important pre-clinical models for stem cell research. Therefore, this study investigated the use of SSBs as an alternative culture method for the expansion of iPSCs. Using an established porcine iPSC line as well as a new cell line derived and characterized in the current study, we report that porcine iPSCs (piPSCs) can grow in SSB while maintaining characteristics of pluripotency and karyotypic stability similar to cells grown in traditional two-dimensional static culture. This culture method provides a suitable platform for scale up of cell culture to provide adequate cell numbers for future research applications involving porcine induced pluripotent stem cells.

Genomic Environments and Their Influence on Transposable Element Communities

Saylor, B. — 2019-06-11

BackgroundDespite decades of research the factors that cause differences in transposable element (TE) distribution and abundance within and between genomes are still unclear. Transposon Ecology is a new field of TE research that promises to aid our understanding of this often-large part of the genome by treating TEs as species within their genomic environment, allowing the use of methods from ecology on genomic TE data. Community ecology methods are particularly well suited for application to TEs as they commonly ask questions about how diversity and abundance of a community of species is determined by the local environment of that community.nnResultsUsing a redundancy analysis, we found that ~ 50% of the TEs within a diverse set of genomes are distributed in a predictable pattern along the chromosome, and the specific TE superfamilies that show these patterns are relate to the phylogeny of the host taxa. In a more focused analysis, we found that ~60% of the variation in the TE community within the human genome is explained by its location along the chromosome, and of that variation two thirds (~40% total) was explained by the 3D location of that TE community within the genome (i.e. what other strands of DNA physically close in the nucleus). Of the variation explained by 3D location half (20% total) was explained by the type of regulatory environment (sub compartment) that TE community was located in. Using an analysis to find indicator species, we found that some TEs could be used as predictors of the environment (sub compartment type) in which they were found; however, this relationship did not hold across different chromosomes.nnConclusionsThese analyses demonstrated that TEs are non-randomly distributed across many diverse genomes and were able to identify the specific TE superfamilies that were non-randomly distributed in each genome. Furthermore, going beyond the one-dimensional representation of the genome as a linear sequence was important to understand TE patterns within the genome. Additionally, we extended the utility of traditional community ecology methods in analyzing patterns of TE diversity.

Variations in terrestrial arthropod DNA metabarcoding methods recovers robust beta diversity but variable richness and site indicators based on exact sequence variants

Porter, T. M. — 2019-07-05

Terrestrial arthropod fauna have been suggested as a key indicator of ecological integrity in forest systems. Because phenotypic identification is expert-limited, a shift towards DNA metabarcoding could improve scalability and democratize the use of forest floor arthropods for biomonitoring applications. The objective of this study was to establish the level of field sampling and DNA extraction replication needed for soil arthropod biodiversity assessments. Processing individually collected field samples recovered significantly higher richness (539-596 ESVs) than pooling the same number of field samples (126-154 ESVs), and we found no significant richness differences when using 1 or 3 pooled DNA extractions. Variations in the number of individual or composite samples or DNA extractions resulted in similar sample clustering based on community dissimilarities. Though our ability to identify taxa to species rank was limited, we were able to use arthropod COI metabarcodes from forest soil to assess richness, distinguish among sites, and recover site indicators based on unnamed exact sequence variants. Our results highlight the need to continue DNA barcoding of local taxa during COI metabarcoding studies to help build reference databases. All together, these sampling considerations support the use of soil arthropod COI metabarcoding as a scalable method for biomonitoring.

A reference library for the identification of Canadian invertebrates: 1.5 million DNA barcodes, voucher specimens, and genomic samples

deWaard, J. R. — 2019-07-14

The reliable taxonomic identification of organisms through DNA sequence data requires a well parameterized library of curated reference sequences. However, it is estimated that just 15% of described animal species are represented in public sequence repositories. To begin to address this deficiency, we provide DNA barcodes for 1,500,003 animal specimens collected from 23 terrestrial and aquatic ecozones at sites across Canada, a nation that comprises 7% of the planets land surface. In total, 14 phyla, 43 classes, 163 orders, 1123 families, 6186 genera, and 64,264 Barcode Index Numbers (BINs; a proxy for species) are represented. Species-level taxonomy was available for 38% of the specimens, but higher proportions were assigned to a genus (69.5%) and a family (99.9%). Voucher specimens and DNA extracts are archived at the Centre for Biodiversity Genomics where they are available for further research. The corresponding sequence and taxonomic data can be accessed through the Barcode of Life Data System, GenBank, the Global Biodiversity Information Facility, and the Global Genome Biodiversity Network Data Portal.nnnnO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/701805v1_ufig1.gif" ALT="Figure 1">nView larger version (17K):norg.highwire.dtl.DTLVardef@5c6449org.highwire.dtl.DTLVardef@1bc0224org.highwire.dtl.DTLVardef@309dddorg.highwire.dtl.DTLVardef@1cc2cc8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Intraperitoneal injection of sodium pentobarbital is associated with pain in rats.

Reimer, J. — 2019-07-15

An effective and pain-free killing method is required to achieve the goal of euthanasia, a "good death". Overdose of sodium pentobarbital (PB) by intraperitoneal (IP) injection is a widely accepted technique, but questions remain regarding pain associated with administration. As PB rapidly causes sedation and loss of consciousness, most studies have relied on indirect evidence of pain. The objective of this study was to assess pain associated with IP PB using an appropriate vehicle control.nnAdult male and female Sprague Dawley (SD) and female Wistar rats (N = 112) were block randomised by sex and strain to receive one of four treatments: 1) 800 mg/kg PB (pH 11); 2) 800 mg/kg PB with 4 mg/kg lidocaine (PB+lido); 3) saline or 4) vehicle controls (pH 11 or 12.5). Behavior (Rat Grimace Scale [RGS], writhing, back arching) was evaluated at baseline, before loss of righting reflex (PB and PB+lido groups), 80s, 151s and 10 min post-injection (PI; saline and vehicle control groups).nnIn the vehicle control groups, the RGS scores were increased at 151s PI (SD: p = 0.0008, 95%CI -0.731 to -0.202) from baseline, as was relative frequency of writhing (SD: p < 0.00001; Wistar; p = 0.0004). RGS scores remained elevated 10 mins PI (SD: p = 0.0070, 95%CI -0.768 to -0.118; Wistar: p = 0.0236, 95%CI -0.907 to -0.0742) but the relative frequency of writhing did not (p > 0.05). The RGS scores and the relative frequency of writhing remained low in the PB, PB+lido and saline groups (p > 0.05). Back arching increased from baseline in the PB+lido group before loss of righting reflex and in the vehicle control group (SD rats) at 151s PI (p < 0.05).nnThese results show that IP PB results in signs associated with pain. The sedative effects of PB limit behavioral assessment.

BIN overlap confirms transcontinental distribution of pest aphids (Hemiptera: Aphididae)

Naseem, M. T. — 2019-07-17

DNA barcoding is highly effective for identifying specimens once a reference sequence library is available for the species assemblage targeted for analysis. Despite the great need for an improved capacity to identify the insect pests of crops, the use of DNA barcoding is constrained by the lack of a well-parameterized reference library. The current study begins to address this limitation by developing a DNA barcode reference library for the pest aphids of Pakistan. It also examines the affinities of these taxa with conspecific taxa from other geographic regions based on both conventional taxonomy and Barcode Index Numbers (BINs). A total of 809 aphids were collected from 123 plant species at 87 sites across Pakistan. Morphological study and DNA barcoding allowed 774 specimens to be identified to one of 42 species while the others were placed to a genus or subfamily. The 801 sequences obtained from these specimens were assigned to 52 BINs whose monophyly were supported by neighbor-joining (NJ) clustering and Bayesian inference. The 42 species were assigned to 41 BINs with 38 showing BIN concordance; one species (Rhopalosiphum padi) was assigned to two BINs, while two others (Aphis affinis, Aphis gossypii) were assigned to the same BIN, while one species (Aphis astragalina) lacked a qualifying sequence. The 42 Linnaean species were represented on BOLD by 7,870 records from 69 countries. Combining these records with those from Pakistan produced to 60 BINs with 12 species showing a BIN split and three a BIN merger. Geo-distance correlations showed that intraspecific divergence values for 18 of 37 species were not affected by the distance between populations. Forty four of the 52 BINs from Pakistan had counterparts in 73 countries across six continents, documenting the broad distributions of pest aphids.

Pre-warming before general anesthesia with isoflurane delays the onset of hypothermia in rats

Rufiange, M. — 2019-07-26

General anesthesia causes hypothermia by impairing normal thermoregulatory mechanisms. Redistribution of warm blood from the core to the periphery is the primary mechanism in the development of hypothermia and begins following induction of anesthesia. Raising skin temperature before anesthesia reduces the temperature gradient between core and periphery, decreasing the transfer of heat. This prospective, crossover study (n = 17 adult male and female SD rats) compared three treatment groups: PW1% (pre-warming to increase core temperature 1% over baseline), PW40 (pre-warming to increase core temperature to 40{degrees}C) and NW (no warming). The PW1% group was completed first to ensure tolerance of pre-warming. Treatment order was then randomized and alternated after a washout period. Once target temperature was achieved, anesthesia was induced and maintained with isoflurane in oxygen without further external temperature support. Pre-warming was effective at delaying the onset of hypothermia, with a significant difference between PW1% (11.2 minutes) and PW40 (14.7 minutes, p = 0.0044 (95%CI -12 to -2.2), PW40 and NW (6.0 minutes, p = 0.003 (95%CI 1.8 to 8.7) and PW1% and PW40 (p = 0.004, 95%CI -12 to -2.2). The rate of heat loss in the pre-warmed groups exceed that of the NW group: PW1% versus NW (p = 0.005, 95%CI 0.004 to 0.027), PW40 versus NW (p < 0.0001, 95%CI 0.014 to 0.036) and PW1% versus PW40 (p = 0.07, 95%CI -0.021 to 0.00066). Pre-warming alone confers a protective effect against hypothermia during volatile anesthesia; however, longer duration procedures would require additional heating support.

Sex differences in innate and adaptive neural oscillatory patterns predict resilience and susceptibility to chronic stress in rats

Theriault, R.-K. — 2019-07-30

Major Depressive Disorder (MDD) is a chronic illness with higher incidence in women. Dysregulated neural oscillatory activity is an emerging mechanism underlying MDD, however whether sex differences in these rhythms contribute to the development of MDD symptoms is unknown. Using the chronic unpredictable stress model, we found that stress-resilient and susceptible animals exhibited sex-specific oscillatory markers in the prefrontal cortex, cingulate cortex, nucleus accumbens and hippocampus. Resilient females were predominantly characterized by increased hippocampal theta power and coherence, while resilient males exhibited increased system-wide gamma coherence. In susceptible animals, the females displayed a widespread increase in delta and reduced theta power, however males showed few within-sex differences that could delineate stress susceptibility from resilience. Finally, stress responses were mediated by the temporal recruitment of specific neural pathways, culminating in system-wide changes that correlated with the expression of depression-like behaviours. These findings show that neurophysiological responses can serve as predictive markers of behaviours linked to depression in a sex-specific manner.

Forensics and DNA Barcodes - Do Identification Errors Arise in the Lab or in the Sequence Libraries?

Pentinsaari, M. — 2019-08-20

Forensic studies often require the determination of biological materials to a species level. As such, DNA-based approaches to identification, particularly DNA barcoding, are attracting increased interest. The capacity of DNA barcodes to assign newly encountered specimens to a species relies upon access to informatics platforms, such as BOLD and GenBank, which host libraries of reference sequences and support the comparison of new sequences to them. As parameterization of these libraries expands, DNA barcoding has the potential to make valuable contributions in diverse forensic contexts. However, a recent publication called for caution after finding that both platforms performed poorly in identifying specimens of 17 common insect species. This study follows up on this concern by asking if the misidentifications reflected problems in the reference libraries or in the query sequences used to test them. Because this reanalysis revealed that missteps in acquiring and analyzing the query sequences were responsible for the misidentifications, a workflow is described to minimize such errors in future investigations. The present study also revealed the limitations imposed by the lack of a polished species-level taxonomy for many groups. In such cases, forensic applications can be strengthened by mapping the geographic distributions of sequence-based species proxies rather than waiting for the maturation of formal taxonomic systems based on morphology.

Electrophysiological correlates of the flexible allocation of visual working memory resources

Salahub, C. — 2019-08-28

Visual working memory is a brief, capacity-limited store of visual information that is involved in a large number of cognitive functions. To guide ones behavior effectively, one must efficiently allocate these limited memory resources across memory items. Previous research has suggested that items are either stored in memory or completely blocked from memory access. However, recent behavioral work proposes that memory resources can be flexibly split across items based on their level of task importance. Here, we investigated the electrophysiological correlates of flexible resource allocation by manipulating the distribution of resources amongst systematically lateralized memory items. We examined the contralateral delay activity (CDA), a waveform typically associated with the number of items held in memory. Across three experiments, we found that, in addition to memory load, the CDA flexibly tracks memory resource allocation. This allocation occurred as early as attentional selection, as indicated by the N2pc. Additionally, CDA amplitude was better-described when fit with a continuous model predicted by load and resources together than when fit with either alone. Our findings show that electrophysiological markers of attentional selection and memory maintenance not only track memory load, but also the proportion of memory resources those items receive.

Variant calling for cpn60 barcode sequence-based microbiome profiling

Vancuren, S. J. — 2019-08-29

Amplification and sequencing of conserved genetic barcodes such as the cpn60 gene is a common approach to determining the taxonomic composition of microbiomes. Exact sequence variant calling has been proposed as an alternative to previously established methods for aggregation of sequence reads into operational taxonomic units (OTU). We investigated the utility of variant calling for cpn60 barcode sequences and determined the minimum sequence length required to provide species-level resolution. Sequence data from the 5 region of the cpn60 barcode amplified from the human vaginal microbiome (n=45), and a mock community were used to compare variant calling to de novo assembly of reads, and mapping to a reference sequence database in terms of number of OTU formed, and overall community composition. Variant calling resulted in microbiome profiles that were consistent in apparent composition to those generated with the other methods but with significant logistical advantages. Variant calling is rapid, achieves high resolution of taxa, and does not require reference sequence data. Our results further demonstrate that 150 bp from the 5 end of the cpn60 barcode sequence is sufficient to provide species-level resolution of microbiota.

Accumulation of genetic variants associated with immunity in the selective breeding of broilers

Zou, A. — 2019-09-12

To satisfy an increasing demand for dietary protein, the poultry industry has employed genetic selection to increase the growth rate of broilers by over 400% in the past 50 years. Although modern broilers reach a marketable weight of [~]2 kg in a short span of 35 days, a speed twice as fast as a broiler 50 years ago, the expedited growth has been associated with several negative detrimental consequences. Aside from heart and musculoskeletal problems, which are direct consequences of additional weight, the immune response is also thought to be altered in modern broilers. Given that identifying the underlying genetic basis responsible for a less sensitive innate immune response would be economically beneficial for poultry breeding, we decided to compare the genomes of two unselected meat control strains that are representative of broilers from 1957 and 1978, and a current commercial broiler line. Through analysis of genetic variants, we developed a custom prioritization strategy to identify genes and pathways that have accumulated genetic changes and are biologically relevant to immune response and growth performance. Our results highlight two genes, TLR3 and PLIN3, with genetic variants that are predicted to enhance growth performance at the expense of immune function. Placing these new genomes in the context of other chicken lines, reveal genetic changes that have specifically arisen in selective breeding programs that were implemented in the last 50 years.

Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster

Whitfield, G. B. — 2019-09-18

Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems (https://doi.org/10.1101/769745). Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3,5-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of Bacilli, Clostridia, Streptococci, and Actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.nnAuthor summaryThe Pel polysaccharide is required for biofilm formation in P. aeruginosa and we have previously found that the genes necessary for biosynthesis of this polymer are broadly distributed across Gram-negative bacteria. Herein, we show that many species of Gram-positive bacteria also possess Pel biosynthetic genes and demonstrate that these genes are used Bacillus cereus for biofilm formation. We show that Pel production in B. cereus is regulated by c-di-GMP and have identified two enzymes, a diguanylate cyclase, CdgF, and a phosphodiesterase, CdgE, that control the levels of this bacterial signalling molecule. While Pel production in B. cereus also requires the binding of c-di-GMP to the receptor PelD, the divergence of this protein in Streptococci suggests a c-di-GMP independent mechanism of regulation is used in this species. The discovery of a Pel biosynthetic gene cluster in Gram-positive bacteria and our characterization of the pel operon in B. cereus suggests that Pel is a widespread biofilm component across all bacteria.

Clinical evaluation of postoperative analgesia and serum biochemical changes of paracetamol compared to meloxicam and carprofen in bitches undergoing ovariohysterectomy

Hernandez -Avalos, I. — 2019-09-27

BackgroundIn veterinary medicine, the administration of nonsteroidal anti-inflammatory analgesics (NSAIDs) for the control of postsurgical pain in dogs and cats is common given the anti-inflammatory, analgesic, and antipyretic effects of these drugs. This study compared the serum biochemical changes and postoperative analgesic effects of paracetamol, meloxicam, and carprofen in bitches submitted to an ovariohysterectomy using the Dynamic Interactive Visual Analog Scale (DIVAS) and Pain Scale of the University of Melbourne (UMPS) scoring systems.nnMethodsThirty bitches of different breeds underwent elective ovariohysterectomies and were randomly assigned to one of three treatment groups: a paracetamol group [15 mg kg-1 intravenous (IV)], a carprofen group (4 mg kg-1 IV), and a meloxicam group (0.2 mg kg-1 IV). All treatments were administered 30 minutes prior to surgery. Paracetamol was administered every 8 hours postoperatively for 48 hours total, while carprofen and meloxicam were intravenously administered every 24 hours. An evaluation of post-surgical pain was done with the DIVAS and the UMPS. The first post-surgical pain measurement was performed 1 hour after surgery and then 2, 4, 6, 8, 12, 16, 20, 24, 36, and 48 hours after surgery.nnResultsAll groups exhibited a gradual reduction in pain throughout the postoperative period in both scales; however, neither scale significantly differed between the three treatment groups (P > 0.05) during the 48 postoperative hours.nnConclusionsParacetamol was as effective as meloxicam and carprofen for post-surgical analgesia in bitches subjected to elective ovariohysterectomy. The present study demonstrates that paracetamol may be considered a tool for the effective treatment of acute perioperative pain in dogs. Furthermore, this drug led to no adverse reactions or changes in the parameters assessed in the present study, indicating its safety.

Validating metabarcoding-based biodiversity assessments with multi-species occupancy models: a case study using coastal marine eDNA

McClenaghan, B. — 2019-10-08

Environmental DNA (eDNA) metabarcoding is an increasingly popular method for rapid biodiversity assessment. As with any ecological survey, false negatives can arise during sampling and, if unaccounted for, lead to biased results and potentially misdiagnosed environmental assessments. We developed a multi-scale, multi-species occupancy model for the analysis of community biodiversity data resulting from eDNA metabarcoding; this model accounts for imperfect detection and additional sources of environmental and experimental variation. We present methods for model assessment and model comparison and demonstrate how these tools improve the inferential power of eDNA metabarcoding data using a case study in a coastal, marine environment. Using occupancy models to account for factors often overlooked in the analysis of eDNA metabarcoding data will dramatically improve ecological inference, sampling design, and methodologies, empowering practitioners with an approach to wield the high-resolution biodiversity data of next-generation sequencing platforms.

Diversity and complexity of the large surface protein family in the compacted genomes of various Pneumocystis species

Ma, L. — 2019-10-25

Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multi-copy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins, and provide detailed description of the classification, unique characteristics and phylogenetic relatedness of five Msg families. We further describe the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests a wide variety of functions for this superfamily, not only conferring antigenic variation to allow immune evasion but also mediating life-stage development, optimizing cell mobility and adhesion, and adapting to specific host niches or environmental conditions. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.

Behavioral and physiological evidence that increasing group size ameliorates the impacts of social disturbance

Anderson, H. M. — 2019-10-25

Intra-group social stability is important for the long-term productivity and health of social organisms. We evaluated the effect of group size on group stability in the face of repeated social perturbations using a cooperatively breeding fish, Neolamprologus pulcher. In a laboratory study, we compared both the social and physiological responses of individuals from small versus large groups to the repeated removal and replacement of the most dominant group member (the breeder male). Individuals living in large groups were overall more resistant to instability but were seemingly slower to recover from perturbation. Members of small group were more vulnerable to instability but recovered faster. Breeder females in smaller groups also showed greater physiological preparedness for instability following social perturbations. In sum, we recover both behavioral and physiological evidence that living in larger groups helps to dampen the impacts of social instability in this system.nnSO_SCPLOWUMMARYC_SCPLOW SO_SCPLOWTATEMENTC_SCPLOWSocial stability is vital for group productivity and long-term persistence. Here, both behavioral and physiological evidence conveys that larger groups are less susceptible to social disturbance.

Metabarcoding a Metacommunity: detecting change in a wetland wilderness

Bush, A. — 2019-10-25

The complexity and natural variability of ecosystems present a challenge for reliable detection of change due to anthropogenic influences. This issue is exacerbated by necessary trade-offs that reduce the quality and resolution of survey data for assessments at large-scales. The Peace-Athabasca Delta (PAD) is a large inland wetland complex in northern Alberta, Canada. Despite its geographic isolation, the PAD is threatened by encroachment of oil sands mining in the Athabasca watershed, and hydroelectric dams in the Peace watershed. Methods capable of reliably detecting changes in ecosystem health are needed to evaluate and manage risks. Between 2011 and 2016, aquatic macroinvertebrates were sampled across a gradient of wetland flood frequency, applying both microscope-based morphological identification, and DNA metabarcoding. Using multi-species occupancy models, we demonstrate that DNA metabarcoding detected a much broader range of taxa and more taxa per sample compared to traditional morphological identification, and was essential to identifying significant responses to flood and thermal regimes. We show that family-level occupancy masks high variation among genera, and for the first time, quantify the bias of barcoding primers on the probability of detection in a natural community. Interestingly, patterns of community assembly were near random, suggesting a strong role of stochasticity in the dynamics of the metacommunity. This variability seriously compromises effective monitoring at local scales, but also reflects resilience to hydrological and thermal variability. Nevertheless, simulations showed the greater efficiency of metabarcoding, particularly at a finer taxonomic resolution, provided the statistical power needed to detect change at the landscape scale.

Identification of the Clostridial cellulose synthase and characterization of the cognate glycosyl hydrolase, CcsZ

Scott, W. — 2019-11-10

Biofilms are community structures of bacteria enmeshed in a self-produced matrix of exopolysaccharides. The biofilm matrix serves numerous roles, including resilience and persistence, making biofilms a subject of research interest among persistent clinical pathogens of global health importance. Our current understanding of the underlying biochemical pathways responsible for biosynthesis of these exopolysaccharides is largely limited to Gram-negative bacteria. Clostridia are a class of Gram-positive, anaerobic and spore-forming bacteria, and include the important human pathogens Clostridium perfringens, Clostridium botulinum, and Clostridioides difficile, among numerous others. Clostridia have been reported to form biofilms composed of cellulose, although the specific loci which encode the cellulose synthase have not been identified. Here, we report the discovery of a gene cluster, which we named ccsABZCD, among selected bacteria within class Clostridia that appears to encode a synthase complex responsible for polymerization, modification, and export of an O-acetylated cellulose exopolysaccharide. To test this hypothesis, we subcloned the putative glycoside hydrolase CcsZ and solved the X-ray crystal structure of both apo- and product-bound CcsZ. Our results demonstrate that CcsZ is in fact an endo-acting cellulase belonging to glycoside hydrolase family 5 (GH-5). This is in contrast to the Gram-negative cellulose synthase, which instead encodes BcsZ, a structurally distinct GH-8. We further show CcsZ is capable of hydrolysis of the soluble mock substrate carboxymethylcellulose (CMC) with a pH optimum of 4.5. The data we present here serves as an entry point to an understanding of biofilm formation among class Clostridia and allowed us to predict a model for Clostridial cellulose synthesis. Author summaryBiofilms are communities of microorganisms that enmesh themselves in a protective matrix of elf-produced polysaccharide materials. Biofilms have demonstrated roles in both virulence and persistence among bacterial pathogens of global health importance. The class Clostridia are a polyphyletic grouping of primarily Gram-positive, anaerobic and spore-forming bacteria which contain the important and well-studied human pathogens Clostridioides difficile, Clostridium botulinum, and Clostridium perfringens, among others. Bacteria belonging to class Clostridia have been anecdotally reported to form biofilms made of cellulose, although the molecular mechanisms governing their production has not before been described. In this work, we identify a gene cluster, which we name ccsABZHI, for the Clostridial cellulose synthase, which bears remarkable similarity to molecular machinery required for the production of cellulose biofilms in other Gram-negative bacteria. We further biochemically characterize one of these enzymes, CcsZ, a predicted endoglucanase which we predicted from our model should cleave cellulose exopolysaccharides. We show that CcsZ is in fact capable of this activity and belongs to a broader family of glycoside hydrolases with unexpected taxonomic diversity. Our work represents an entry point towards an understanding of the molecular mechanisms governing cellulose biofilm formation in Gram-positive bacteria.

Winter in water: Differential responses and the maintenance of biodiversity

McMeans, B. — 2019-11-21

The ecological consequences of winter in freshwater systems are an understudied but rapidly emerging research area. Here, we argue that winter periods of reduced temperature and light (and potentially oxygen and resources) could play an underappreciated role in mediating the coexistence of species. This may be especially true for temperate and subarctic lakes, where seasonal changes in the thermal environment might fundamentally structure species interactions. With climate change already shortening ice-covered periods on temperate and polar lakes, consideration of how winter conditions shape biotic interactions is urgently needed. Using freshwater fishes in northern temperate lakes as a case study, we demonstrate how physiological trait differences (e.g., thermal preference, light sensitivity) drive differential behavioral responses to winter among competing species. Specifically, some species have a higher capacity for winter activity than others. Existing and new theory is presented to argue that such differential responses to winter can promote species coexistence. Importantly, if winter is a driver of niche differences that weaken competition between relative to within species, then shrinking winter periods could threaten coexistence by tipping the scales in favor of certain sets of species over others.

Impacts of intraspecific variability and local adaptation on the ecophysiology of mosses: an example with Sphagnum magellanicum

Oke, T. A. — 2019-11-29

BackgroundBryophytes are a diverse plant group and are functionally different from vascular plants. Yet, plant ecology theories and hypotheses are often presented in an inclusive term. The trait-based approach to ecology is no exception; largely focusing on vascular plant traits and almost exclusively on interspecific traits. Currently, we lack information about the magnitude and the importance of intraspecific variability to the ecophysiology of bryophytes and how these might translate to local adaptation--a prerequisite for adaptive evolution. MethodWe used transplant and factorial experiments involving moisture and light to ask whether variability in traits between morphologically distinct individuals of Sphagnum magellanicum from habitat extremes was due to phenotypic plasticity or local adaptation and the implications for the ecophysiology of the species. Key ResultsWe found that the factors that discriminated between the plant origins in the field did not translate to their ecophysiological functioning and the pattern of variability changed with the treatments, which suggests that the trait responses were due largely to phenotypic plasticity. The trait responses suggest that the need for mosses to grow in clumps where they maintain a uniform growth rate may have an overriding effect on responses to environmental heterogeneity, and therefore a constraint for local adaptation. ConclusionThe circumstances under which local adaptation would be beneficial in this plant group is not clear. We conclude that extending the trait-based framework to mosses or making comparisons between mosses and vascular plants under any theoretical framework would only be meaningful to the extent that growth form and dispersal strategies are considered.

Invasive plants as novel food resources, the pollinators’ perspective

Ignasi Bartomeus — 2015-05-05

Biological invasions are one of the main drivers of global change and have negatively impacted all biomes and trophic levels (Hobbs 2000, Vila et al. 2011). While most introduced species fail to establish, or establish small naturalized populations (hereafter exotic species), a few become invasive and rapidly increase in abundance and/or range (hereafter invasives or invaders; Pysek et al. 2004). It is these invader species that are most often linked to negative impacts on native / endemic communities. Although most interactions between invasive and native species at the same trophic level result in negative direct impacts (e.g. plant-plant competitive interactions, Vila et al. 2011), when the invasive plant species can be used as a resource those interactions can also be positive for consumers such as native herbivores, predators or mutualists, at least for some species (Heleno et al. 2009, Bezemer et al. 2004). Entomophilous exotic plant species, for example, not only compete directly for space and light with other plants, but also offer resource opportunities for the native pollinator community (Stouffer et al. 2014). Most research on this topic to date has taken the plant perspective, focusing on how successful plant invaders integrate into the native plant-pollinator interaction networks (Vila et al. 2009), and how this integration in turn impacts the native plant community (Morales and Traveset 2009). However, species specific responses of pollinators to the addition of exotic plants are rarely taken into account. This represent an important gap in our knowledge as pollinator foraging choices determine the structure of interactions within communities, which in turn have important implications for the community stability (Thebault and Fontain 2010) and functioning (Thomson et al. 2012). How different pollinators respond to the changed composition of floral species within the community that results from exotic plant invaders deserves more attention.

Single cell chemotactic responses of Helicobacter pylori to urea in a microfluidic chip

Xuan Weng — 2016-03-23

Helicobacter pylori is a spiral-shaped bacterium that grows in the human digestive tract; it infects ~50% of the global population. H. pylori induce inflammation, gastroenteritis, and ulcers, which is associated with significant morbidity and may be linked to stomach cancer in certain individuals. Motility is an essential virulence factor for H. pylori, allowing it to migrate toward and invade the epithelial lining of the stomach to shelter it from the harsh environment of the stomach. H. pylori senses pH gradients and use polar flagella to move towards the epithelium where the pH approaches neutrality. However, its chemotaxis behaviors are incompletely understood. Previous in vitro tests examining the response of H. pylori to chemical gradients have been subjected to substantial limitations. To more accurately mimic/modulate the cellular microenvironment, a nanoporous microfluidic device was used to monitor the real time chemotactic activity of single cell of H. pylori in response to urea. The results showed that microfluidic method is a promising alternative for precisely studying chemotactic behavior of bacteria.

Environmental DNA Barcode Sequence Capture: Targeted, PCR-free Sequence Capture for Biodiversity Analysis from Bulk Environmental Samples

Shokralla, S. — 2016-11-13

Environmental DNA analysis using PCR amplified marker genes has been a key application of high-throughput sequencing (HTS). However, PCR bias is a major drawback to gain accurate qualitative and quantitative biodiversity data. We developed a PCR-free approach using enrichment baits for species-specific mitochondrial cytochrome c oxidase 1(COI) DNA barcodes. The sequence capture was tested on species-rich bulk terrestrial and aquatic benthic samples. Hybridization capture recovered an average of 6 and 4.7 more arthropod orders than amplicon sequencing for terrestrial and benthic samples, respectively. For the terrestrial sample, the four most abundant arthropod orders comprised 94.0% of the sample biomass. These same four orders comprised 95.5% and 97.5% of the COI sequences recovered by amplification and capture, respectively. Hybridization capture recovered three arthropod orders that were detected by biomass analysis, but not by amplicon sequencing and two other insect orders that were not detected by either biomass or amplicon methods. These results indicate the advantage of using sequence capture for a more accurate analysis of biodiversity in bulk environmental samples. The protocol can be easily customized to other DNA barcode markers or gene regions of interest for a wide range of taxa or for a specific target group.

Local differentiation in the defensive morphology of an invasive zooplankton species is not genetically based

Fiorino, G. E. — 2017-01-06

Evolutionary changes in functional traits represent one possible reason why exotic species spread to become invasive, but empirical studies of the mechanisms driving phenotypic differentiation between populations of invasive species are rare. This study tested whether differences in distal spine length among populations of the invasive cladoceran, Bythotrephes longimanus, could be explained by local adaptation or phenotypic plasticity. We collected Bythotrephes from six lakes and found that distal spine lengths and natural selection on distal spine length differed among populations, but were unrelated to the gape-limitation of the dominant fish predator in the lake from which they were collected. A common garden experiment revealed significant genetic and maternal variation for distal spine length, but phenotypic differences among populations were not genetically based. Phenotypic differences among lakes in this ecologically important trait are, therefore, the result of plasticity and not local adaptation, despite spatially variable selection on this heritable trait. The ability of Bythotrephes to plastically adjust distal spine length may explain the success of this species at invading lake ecosystems with diverse biotic environments.

Comparing three types of dietary samples for prey DNA decay in an insect generalist predator

Kamenova, S. — 2017-01-06

The rapidly growing field of molecular diet analysis is becoming increasingly popular among ecologists, especially when investigating methodologically challenging groups such as invertebrate generalist predators. Prey DNA detection success is known to be affected by multiple factors, however the type of dietary sample has rarely been considered. Here, we address this knowledge gap by comparing prey DNA detection success from three types of dietary samples. In a controlled feeding experiment, using the carabid beetle Pterostichus melanarius as a model predator, we collected regurgitates, feces and whole consumers (including their gut contents) at different time points post-feeding. All dietary samples were analyzed using multiplex PCR targeting three different length DNA fragments (128 bp, 332 bp and 612 bp). Our results show that both the type of dietary sample and the size of the DNA fragment contribute to a significant part of the variation found in the detectability of prey DNA. Specifically, we observed that in both regurgitates and whole consumers prey DNA was detectable significantly longer for all fragment sizes than for feces. Based on these observations, we conclude that prey DNA detected from regurgitates and whole consumers DNA extracts are comparable, whereas prey DNA detected from feces, though still sufficiently reliable for ecological studies, will not be directly comparable to the former. Therefore, regurgitates and feces constitute an interesting, non-lethal source for dietary information that could be applied to field studies in situations when invertebrate predators should not be killed.

Environmental biases in the study of ecological networks at the planetary scale

Poisot, T. — 2020-01-28

Ecological networks are increasingly studied at large spatial scales, expanding their focus from a conceptual tool for community ecology into one that also adresses questions in biogeography and macroecology. This effort is supported by increased access to standardized information on ecological networks, in the form of openly accessible databases. Yet, there has been no systematic evaluation of the fitness for purpose of these data to explore synthesis questions at very large spatial scales. In particular, because the sampling of ecological networks is a difficult task, they are likely to not have a good representation of the diversity of Earths bioclimatic conditions, likely to be spatially aggregated, and therefore unlikely to achieve broad representativeness. In this paper, we analyze over 1300 ecological networks in the mangal.io database, and discuss their coverage of biomes, and the geographic areas in which there is a deficit of data on ecological networks. Taken together, our results suggest that while some information about the global structure of ecological networks is available, it remains fragmented over space, with further differences by types of eco-logical interactions. This causes great concerns both for our ability to transfer knowledge from one region to the next, but also to forecast the structural change in networks under climate change.

Chronic Chemogenetic Manipulation of Ventral Pallidum Targeted Neurons in Rats Fed an Obesogenic Diet.

Doucette, W. T. — 2020-02-19

Current treatments for obesity are unable to reliably reduce weight over time and alternative treatments need to address this challenge. New interventions that target the nervous system could manipulate brain networks underlying reward, metabolic rate and behavior. Here, the ventral pallidum was evaluated as a target of manipulation due to its established role in the networks underlying motivation, pleasure and behavioral output. Chronic inhibitory or excitatory chemogenetic activation was used to modulate the activity of ventral pallidum (VP) targeted neurons in rats on an obesogenic diet. We hypothesized that inhibition of VP activity would decrease the salience of the rats high-sugar, high-fat diet and lead to reduced food consumption and weight gain over time. Paradoxically, measurements of weight, water and food consumption revealed significantly increased weight gain in both groups receiving VP targeted manipulation that was not readily explained by food or water consumption. We theorize that the complex reciprocal feedback between ventral striatal structures (e.g., VP) and metabolic centers of the hypothalamus and brainstem, demonstrated by prior research, likely underpin our findings. This study suggests that the treatment of appetitive disorders (e.g., obesity) with chronic neuromodulation-based interventions could be burdened by the delayed onset of outcomes that are difficult to predict from related prior studies that used acute interventions.

Community-wide seed dispersal distances peak at low levels of specialisation in size-structured networks

Sorensen, M. C. — 2020-02-25

Network approaches provide insight into the complex web of interspecific interactions that structure ecological communities. However, because data on the functional outcomes of ecological networks are very rarely available, the effect of network structure on ecosystem functions, such as seed dispersal, is largely unknown. Here, we develop a new approach that is able to link interaction networks to a trait-based seed-dispersal model to estimate community-wide seed dispersal distances. We simulated networks, using a niche model based on size-matching between plants and birds, that varied in the degree of niche partitioning. We found that community-wide dispersal distances were longest when networks had low degrees of niche partitioning. We further found that dispersal distances of plant species with small fruits peaked in models without niche partitioning, whereas dispersal distances of medium and large-fruited plants peaked at low degrees of niche partitioning. Our simulations demonstrate that the degree of niche partitioning between species is an important determinant of the ecological functions derived from ecological networks and that simulation approaches can provide new insights into the relationship between the structural and functional components of ecological networks.

The utility of a metagenomics approach for marine biomonitoring

Singer, G. A. C. — 2020-03-18

1The isolation and analysis of environmental DNA (eDNA) for ecosystem assessment and monitoring has become increasingly popular. A majority of studies have taken a metabarcoding approach--that is, amplifying and sequencing one or more gene targets of interest. Shotgun sequencing of eDNA--also called metagenomics--while popular in microbial community analysis has not seen much adoption for the analysis of other groups of organisms. Especially in light of the existence of extremely high-capacity DNA sequencers, we decided to test the performance of a shotgun approach side-by-side with a metabarcoding approach on marine water samples obtained from offshore Newfoundland. We found that metabarcoding remains the most efficient technique, but that metagenomics also has significant power to reveal biodiversity patterns, and in fact can be treated as an independent confirmation of ecological gradients. Moreover, we show that metagenomics can also be used to infer factors related to ecosystem health and function.

Improved status and trend estimates from the North American Breeding Bird Survey using a hierarchical Bayesian generalized additive model

Smith, A. C. — 2020-03-29

The status and trend estimates derived from the North American Breeding Bird Survey (BBS), are critical sources of information for bird conservation. However, the estimates are partly dependent on the statistical model used. Therefore, multiple models are useful because not all of the varied uses of these estimates (e.g. inferences about long-term change, annual fluctuations, population cycles, recovery of once declining populations) are supported equally well by a single statistical model. Here we describe Bayesian hierarchical generalized additive models (GAM) for the BBS, which share information on the pattern of population change across a species range. We demonstrate the models and their benefits using data a selection of species; and we run a full cross-validation of the GAMs against two other models to compare predictive fit. The GAMs have better predictive fit than the standard model for all species studied here, and comparable predictive fit to an alternative first difference model. In addition, one version of the GAM described here (GAMYE) estimates a population trajectory that can be decomposed into a smooth component and the annual fluctuations around that smooth. This decomposition allows trend estimates based only on the smooth component, which are more stable between years and are therefore particularly useful for trend-based status assessments, such as those by the IUCN. It also allows for the easy customization of the model to incorporate covariates that influence the smooth component separately from those that influence annual fluctuations (e.g., climate cycles vs annual precipitation). For these reasons and more, this GAMYE model is a particularly useful model for the BBS-based status and trend estimates. LAY SUMMARYO_LIThe status and trend estimates derived from the North American Breeding Bird Survey are critical sources of information for bird conservation, but they are partly dependent on the statistical model used. C_LIO_LIWe describe a model to estimate population status and trends from the North American Breeding Bird Survey data, using a Bayesian hierarchical generalized additive mixed-model that allows for flexible population trajectories and shares information on population change across a species range. C_LIO_LIThe model generates estimates that are broadly useful for a wide range of common conservation applications, such as IUCN status assessments based on trends or changes in the rates of decline for species of concern; and the estimates have better or similar predictive accuracy to other models., and C_LI

Half of the world's tree biodiversity is unprotected and is increasingly threatened by human activities

Guo, W.-Y. — 2020-04-23

Although trees are key to ecosystem functioning, many forests and tree species across the globe face strong threats. Preserving areas of high biodiversity is a core priority for conservation; however, different dimensions of biodiversity and varied conservation targets make it difficult to respond effectively to this challenge. Here, we (i) identify priority areas for global tree conservation using comprehensive coverage of tree diversity based on taxonomy, phylogeny, and functional traits; and (ii) compare these findings to existing protected areas and global biodiversity conservation frameworks. We find that ca. 51% of the top-priority areas for tree biodiversity are located in current protected areas. The remaining half top-priority areas are subject to moderate to high human pressures, indicating conservation actions are needed to mitigate these human impacts. Our findings emphasize the effectiveness of using tree conservation priority areas for future global conservation planning.

A validation scale to determine the readiness of environmental DNA assays for routine species monitoring

Thalinger, B. — 2020-04-29

The use of environmental DNA (eDNA) analysis for species monitoring requires rigorous validation - from field sampling to the analysis of PCR-based results - for meaningful application and interpretation. Assays targeting eDNA released by individual species are typically validated with no predefined criteria to answer specific research questions in one ecosystem. Hence, the general applicability of assays as well as associated uncertainties and limitations, often remain undetermined. The absence of clear guidelines for assay validation prevents targeted eDNA assays from being incorporated into species monitoring and policy; thus, their establishment is essential for realizing the potential of eDNA-based surveys. We describe the measures and tests necessary for successful validation of targeted eDNA assays and the associated pitfalls to form the basis of guidelines. A list of 122 variables was compiled, consolidated into 14 thematic blocks, (e.g. "in silico analysis"), and arranged on a 5-level validation scale from "incomplete" to "operational" with defined minimum validation criteria for each level. These variables were evaluated for 546 published single-species assays. The resulting dataset was used to provide an overview of current validation practices and test the applicability of the validation scale for future assay rating. Of the 122 variables, 20% to 76% were reported; the majority (30%) of investigated assays were classified as Level 1 (incomplete), and 15% did not achieve this first level. These assays were characterised by minimal in silico and in vitro testing, but their share in annually published eDNA assays has declined since 2014. The meta-analysis demonstrates the suitability of the 5-level validation scale for assessing targeted eDNA assays. It is a user-friendly tool to evaluate previously published assays for future research and routine monitoring, while also enabling the appropriate interpretation of results. Finally, it provides guidance on validation and reporting standards for newly developed assays.

The Red Queen's Crown: an evolutionary arms race between coronaviruses and mammalian species reflected in positive selection of the ACE2 receptor among many species

Hajibabaei, M. — 2020-05-15

The world is going through a global viral pandemic with devastating effects on human life and socioeconomic activities. This pandemic is the result of a zoonotic coronavirus, Severe Acute Respirsatory Syndrom Coronavirus 2 (SARS-CoV-2) which is believed to have originated in bats and transferred to humans possibly through an intermediate host species (Zhou et al. 2020; Coronaviridae Study Group of the International Committee on Taxonomy of Viruses 2020). The virus attacks host cells by attaching to a cell membrane surface protein receptor called ACE2 (Ge et al. 2013; Zhou et al. 2020). Given the critical role of ACE2 as a binding receptor for a number of coronaviruses, we studied the molecular evolution of ACE2 in a diverse range of mammalian species. Using ACE2 as the target protein, we wanted to specifically test the Red Queen hypothesis (Dawkins and Krebs 1979) where the parasite and host engage in an evolutionary arms race which can result in positive selection of their traits associated to their fitness and survival. Our results clearly show a phylogenetically broad evolutionary response, in the form of positive selection detected at the codon-level in ACE2. We see positive selection occurring at deep branches as well as 13 incidents at the species level. We found the strongest level of positive selection in Tasmanian devil (Sarcophilus harrisii), donkey (Equus asinus), large flying fox (Pteropus vampyrus), Weddell seal (Leptonychotes weddellii), and dog (Canis lupus familiaris). At the codon-level, we found up to 10% of ACE2 codons are impacted by positive selection in the mammalian lineages studied. This phylogenetically broad evolutionary arms race can contribute to the emergence of new strains of coronaviruses in different mammalian lineages with a potential to transfer between species given the common binding receptor ACE2. Our study provides a molecular evolutionary perspective to the current pandemic and sheds light on its evolutionary mechanisms. "Nothing in biology makes sense except in the light of evolution" (Theodosius Dobzhansky)

A workflow for accurate metabarcoding using nanopore MinION sequencing

Baloglu, B. — 2020-05-25

Metabarcoding has become a common approach to the rapid identification of the species composition in a mixed sample. The majority of studies use established short-read high-throughput sequencing platforms. The Oxford Nanopore MinION, a portable sequencing platform, represents a low-cost alternative allowing researchers to generate sequence data in the field. However, a major drawback is the high raw read error rate that can range from 10% to 22%. To test if the MinION represents a viable alternative to other sequencing platforms we used rolling circle amplification (RCA) to generate full-length consensus DNA barcodes (658bp of cytochrome oxidase I - COI) for a bulk mock sample of 50 aquatic invertebrate species. By applying two different laboratory protocols, we generated two MinION runs that were used to build consensus sequences. We also developed a novel Python pipeline, ASHURE, for processing, consensus building, clustering, and taxonomic assignment of the resulting reads. We were able to show that it is possible to reduce error rates to a median accuracy of up to 99.3% for long RCA fragments (>45 barcodes). Our pipeline successfully identified all 50 species in the mock community and exhibited comparable sensitivity and accuracy to MiSeq. The use of RCA was integral for increasing consensus accuracy, but it was also the most time-consuming step during the laboratory workflow and most RCA reads were skewed towards a shorter read length range with a median RCA fragment length of up to 1262bp. Our study demonstrates that Nanopore sequencing can be used for metabarcoding but we recommend the exploration of other isothermal amplification procedures to improve consensus length.

Examining interspecific density-dependent dispersal in forest small mammals

Denomme-Brown, S. T. — 2020-05-23

The effects of conspecific densities on dispersal have been well documented. However, while positive and negative density-dependent dispersal based on conspecific densities are often shown to be the result of intraspecific competition or facilitation respectively, the effects of heterospecific densities on dispersal are examined far less frequently. This despite the potential for the analogous processes of interspecific competition and heterospecific attraction to influence dispersal. Here we use 51 years of live-trapping data on deer mouse (Peromsycus maniculatus), eastern chipmunk (Tamias striatus), red-backed vole (Myodes gapperi), and jumping mice (Napaeozapus insignis and Zapus hudsonius) to examine the effects of both conspecific and heterospecific densities on dispersal frequency. In terms of conspecific densities, jumping mice were more likely to disperse from areas of low conspecific densities, while red-backed voles and chipmunks did not respond to conspecific densities in their dispersal frequencies. When considering interspecific density effects, while there were no statistically clear effects of density on dispersal frequency, the effects of chipmunk and jumping mice densities on dispersal by red-backed vole were greater than the effects of conspecific densities, with voles more likely to disperse from areas of high chipmunk densities and low jumping mice densities. Likewise, the effect of chipmunk densities on dispersal by jumping mice was greater than the conspecific density effect. Conspecific densities clearly can affect dispersal by two of these four species, but the effects of heterospecific densities on dispersal frequency are less clear. Based on effect sizes it appears that there is potential for heterospecific effects on dispersal by some species in the community, but future experimental work could elucidate the strength and causes of these relationships.

Lateral and longitudinal fish eDNA distribution in dynamic riverine habitats

Thalinger, B. — 2020-05-30

Assessing the status and distribution of fish populations in rivers is essential for management and conservation efforts in these dynamic habitats. Currently, methods detecting environmental DNA (eDNA) are being established as an alternative and/or complementary approach to the traditional monitoring of fish species. In lotic systems, a sound understanding of hydrological conditions and their influence on the local target detection probability and DNA quantity is key for the interpretation of eDNA-based results. However, the effect of seasonal and diurnal changes in discharge and the comparability of semi-quantitative results between species remain hardly addressed. We conducted a cage experiment with four fish species (three salmonid and one cyprinid species) in a glacier-fed, fish-free river in Tyrol (Austria) during summer, fall, and winter discharge situations (i.e. 25-fold increase from winter to summer). Each season, water samples were obtained on three consecutive days at 13 locations downstream of the cages including lateral sampling every 1-2 m across the wetted width. Fish eDNA was quantified by species-specific endpoint PCR followed by capillary electrophoresis. Close to the cages, lateral eDNA distribution was heterogenous and mirrored cage placement within the stream. In addition to the diluting effect of increased discharge, longitudinal signal changes within the first 20 m were weakest at high discharge. For downstream locations with laterally homogenous eDNA distribution, the signals decreased significantly with increasing distance and discharge. Generally, the eDNA of the larger-bodied salmonid species was less frequently detected, and signal strengths were lower compared to the cyprinid species. This study exemplifies the importance of hydrological conditions for the interpretation of eDNA-based data across seasons. To control for heterogenous eDNA distribution and enable comparisons over time, sampling schemes in lotic habitats need to incorporate hydrological conditions and species traits.

Paleoclimate and current climate collectively shape the phylogenetic and functional diversity of trees worldwide

Guo, W.-Y. — 2020-06-03

Trees are of vital importance for ecosystem functioning and services at local to global scales, yet we still lack a detailed overview of the global patterns of tree diversity and the underlying drivers, particularly the imprint of paleoclimate. Here, we present the high-resolution (110 km) worldwide mapping of tree species richness, functional and phylogenetic diversities based on [~]7 million quality-assessed occurrences for 46,752 tree species (80.5% of the estimated total number of tree species), and subsequent assessments of the influence of paleo-climate legacies on these patterns. All three tree diversity dimensions exhibited the expected latitudinal decline. Contemporary climate emerged as the strongest driver of all diversity patterns, with Pleistocene and deeper-time (>107 years) paleoclimate as important co-determinants, and, notably, with past cold and drought stress being linked to reduced current diversity. These findings demonstrate that tree diversity is affected by paleoclimate millions of years back in time and highlight the potential for tree diversity losses from future climate change.

VIRIDIC - a novel tool to calculate the intergenomic similarities of prokaryote-infecting viruses

Moraru, C. — 2020-07-05

Nucleotide based intergenomic similarities are useful to understand how viruses are related with each other and to classify them. Here we have developed VIRIDIC, which implements the traditional algorithm used by the International Committee on Taxonomy of Viruses (ICTV), Bacterial and Archaeal Viruses Subcommittee, to calculate virus intergenomic similarities. When compared with other software, VIRIDIC gave the best agreement with the traditional algorithm. Furthermore, it proved best at estimating the relatedness between more distantly related phages, relatedness that other tools can significantly overestimate. In addition to the intergenomic similarities, VIRIDIC also calculates three indicators of the alignment ability to capture the relatedness between viruses: the aligned fractions for each genome in a pair and the length ratio between the two genomes. The main output of VIRIDIC is a heatmap integrating the intergenomic similarity values with information regarding the genome lengths and the aligned genome fraction. VIRIDIC is available at viridic.icbm.de, both as a web-service and a stand-alone tool. It allows fast analysis of large phage genome datasets, especially in the stand-alone version, which can be run on the user’s own servers and can be integrated in bioinformatics pipelines. VIRIDIC was developed having viruses of Bacteria and Archaea in mind, however, it could potentially be used for eukaryotic viruses as well, as long as they are monopartite.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Harnessing the power of eDNA metabarcoding for the detection of deep-sea fishes

Beverly McClenaghan — 2020-07-12

The deep ocean is the largest biome on Earth and faces increasing anthropogenic pressures from climate change and commercial fisheries. Our ability to sustainably manage this expansive habitat is impeded by our poor understanding of its inhabitants and by the difficulties in surveying and monitoring these areas. Environmental DNA (eDNA) metabarcoding has great potential to improve our understanding of this region and to facilitate monitoring across a broad range of taxa. Here, we evaluate two eDNA sampling protocols and seven primer sets for elucidating fish diversity from deep sea water samples. We found that deep sea water samples (> 1400 m depth) had significantly lower DNA concentrations than surface or mid-depth samples necessitating a refined protocol with a larger sampling volume. We recovered significantly more DNA in large volume water samples (1.5 L) filtered at sea compared to small volume samples (250 mL) held for lab filtration. Furthermore, the number of unique sequences (exact sequence variants; ESVs) recovered per sample was higher in large volume samples. Since the number of ESVs recovered from large volume samples was less variable and consistently high, we recommend the larger volumes when sampling water from the deep ocean. We also identified three primer sets which detected the most fish taxa but recommend using multiple markers due the variability in detection probabilities and taxonomic resolution among fishes for each primer set. Overall, fish diversity results obtained from metabarcoding were comparable to conventional survey methods. While eDNA sampling and processing need be optimized for this unique environment, the results of this study demonstrate that eDNA metabarcoding can be employed to facilitate biodiversity surveys in the deep ocean, require less dedicated survey effort per unit identification and are capable of simultaneously providing valuable information on other taxonomic groups.

Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions

Thalinger, B. — 2020-10-25

The use of sensitive methods is key for the detection of target taxa, from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capillary electrophoresis (celPCR) potentially embodies a viable alternative as it quantitatively measures signal strength in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex PCRs, enabling the simultaneous detection of several target taxa. Here, we compared the sensitivity of singleplex and multiplex celPCR to dPCR for species-specific primer pairs amplifying mitochondrial DNA (COI) of fish species occurring in European freshwaters by analysing dilution series of DNA extracts and field-collected water samples. Both singleplex and multiplex celPCR and dPCR displayed comparable sensitivity with reliable positive amplifications starting at two to 10 target DNA copies per {micro}l DNA extract. celPCR was suitable for quantifying target DNA and direct inference of DNA concentrations from RFU was possible after accounting for primer effects. Furthermore, multiplex celPCRs and dPCRs were successfully used for the detection and quantification of fish-eDNA in field-collected water samples, confirming the results of the dilution series experiment and exemplifying the high sensitivity of the two approaches. The possibility of detection and quantification via multiplex celPCR is appealing for the cost-efficient screening of high sample numbers. The present results confirm the sensitivity of this approach thus enabling its application for future eDNA-based monitoring efforts.

Activity, energy use and species identity affect eDNA shedding in freshwater fish

Thalinger, B. — 2020-10-28

The quantitative measurement of environmental DNA (eDNA) from field-collected water samples is gaining importance for the monitoring of fish communities and populations. The interpretation of these signal strengths depends, among other factors, on the amount of target eDNA shed into the water. However, shedding rates are presumably associated with species-specific traits such as physiology and behavior. Although such differences between juvenile and adult fish have been previously detected, the general impact of movement and energy use in a resting state on eDNA release into the surrounding water remains hardly addressed. In an aquarium experiment, we compared eDNA shedding between seven fish species occurring in European freshwaters. The investigated salmonids, cyprinids, and sculpin exhibit distinct adaptions to microhabitats, diets, and either solitary or schooling behavior. The fish were housed in aquaria with constant water flow and their activity was measured by snapshots taken every 30 s. Water samples for eDNA analysis were taken every 3 h and energy use was determined in an intermittent flow respirometer. After controlling for the effect of fish mass, our results demonstrate a positive correlation between target eDNA quantities as measured with digital PCR, fish activity, and energy use, as well as species-specific differences. For cyprinids, the model based on data from individual fish was only partly transferable to groups, which exhibited lower activity and higher energy use. Our findings highlight the importance of fish physiology and behavior for the comparative interpretation of taxon-specific eDNA quantities. Species traits should therefore be incorporated into eDNA-based monitoring and conservation efforts.

Quaternary climate change explains global patterns of tree beta-diversity

Xu, W.-B. — 2020-11-17

Both historical and contemporary environmental conditions determine present biodiversity patterns, but their relative importance is not well understood. One way to disentangle their relative effects is to assess how different dimensions of beta-diversity relate to past climatic changes, i.e., taxonomic, phylogenetic and functional compositional dissimilarity, and their components generated by replacement of species, lineages and traits (turnover) and richness changes (nestedness). Here, we quantify global patterns of each of these aspects of beta-diversity among neighboring sites for angiosperm trees using the most extensive global database of tree species-distributions (43,635 species). We found that temperature change since the Last Glacial Maximum (LGM) was the major influence on both turnover and nestedness components of beta-diversity, with a negative correlation to turnover and a positive correlation to nestedness. Moreover, phylogenetic and functional nestedness was higher than expected from taxonomic beta-diversity in regions that experienced large temperature changes since the LGM. This pattern reflects relatively greater losses of phylogenetic and functional diversity in species-poor assemblages, possibly caused by phylogenetically and functionally selective species extinction and recolonization during glacial-interglacial oscillations. Our results send a strong warning that rapid anthropogenic climate change is likely to result in a long-lasting phylogenetic and functional compositional simplification, potentially impairing forest ecosystem functioning.

Species heterogeneity in food webs can reduce the potential for alternative stable states

Karatayev, V. A. — 2020-11-27

Alternative stable ecosystem states are possible under the same environmental conditions in many models of 2-3 interacting species and an array of feedback loops. However, multi-species food webs might dissipate the feedbacks that create alternative stable states through species-specific traits and feedbacks. To test this potential, we develop a manyspecies model of consumer-resource interactions with two classes of feedbacks: specialized feedbacks where individual resources become unpalatable at high abundance, or aggregate feedbacks where overall resource abundance reduces consumer recruitment. We quantify how trophic interconnectedness and species differences in demography affect the potential for either feedback to produce alternative stable states dominated by consumers or resources. We find that alternative stable states are likely to happen in many-species food webs when aggregate feedbacks or lower species differences increase redundancy in species contributions to persistence of the consumer guild. Conversely, specialized palatability feedbacks with distinctive species roles in consumer guild persistence reduce the potential for alternative states but increase the likelihood that losing vulnerable consumers cascades into a food web collapse at low stress levels, a dynamic absent in few-species models. Altogether, among-species trait variation can limit the set of processes that create alternative stable states and impede consumer recovery from disturbance.

Defense by duplication: The relation between phenotypic glyphosate resistance andEPSPS gene copy number variation in Amaranthus palmeri

Yakimowski, S. B. — 2021-04-13

Gene copy number variation (CNV) has been increasingly associated with organismal responses to environmental stress, but we know little about the quantitative relation between CNV and phenotypic variation. In this study we quantify variation in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) copy number using digital drop PCR and variation in phenotypic glyphosate resistance in 22 populations of Amaranthus palmeri (Palmer Amaranth), a range-expanding agricultural weed. Overall, we detected a significant positive relation between population mean copy number and mean resistance. The majority of populations exhibited high glyphosate resistance, yet maintained low-resistance individuals resulting in bimodality in many populations. We investigated linear and threshold models for the relation between copy number and resistance, and found evidence for a threshold of ~15 EPSPS copies: there was a steep increase in resistance before the threshold, followed by a much shallower slope. Moreover, as copy number increases, the range of variation in resistance decreases. This suggests a working hypothesis that as EPSPS copies and dosage increases, negative epistatic interactions may be compensated. We detected a quadratic relation between mean resistance and variation (s.d.) in resistance, consistent with the prediction that as phenotypic resistance increases in populations, stabilizing selection decreases variation in the trait. Finally, patterns of variation across the landscape are consistent with less variation among populations in mean copy number / resistance in Georgia where glyphosate resistance was first detected, and wider variation among populations in resistance and copy number in a more northern state where resistance evolution may be at a younger evolutionary state.

Maintaining grass coverage increases methane uptake in Amazonian pasture soils

de Souza, L. F. — 2021-04-26

Cattle ranching is the largest driver of deforestation in the Brazilian Amazon. The rainforest- to-pasture conversion affects the methane cycle in upland soils, changing it from sink to source of atmospheric methane. However, it remains unknown if management practices could reduce the impact of land-use on methane cycling. In this work, we evaluated how pasture management can regulate the soil methane cycle either by maintaining continuous grass coverage on pasture soils, or by liming the soil to amend acidity. Methane fluxes from forest and pasture soils were evaluated in moisture-controlled greenhouse experiments with and without grass cover (Urochloa brizantha cv. Marandu) or liming. In parallel, we assessed changes in the soil microbial community structure of both bare pasture soil as well as rhizosphere soil through high throughput sequencing of the 16S rRNA gene, and quantified the methane cycling microbiota by their respective marker genes related to methane generation (mcrA) or oxidation (pmoA). The experiments used soils from eastern and western Amazonia, and concurrent field studies allowed us to confirm greenhouse data. The presence of a grass cover not only increased methane uptake by up to 35% in pasture soils, but also reduced the abundance of the methane-producing community. In the grass rhizosphere this reduction was up to 10-fold. Methane-producing archaea belonged to the genera Methanosarcina sp., Methanocella sp., Methanobacterium sp., and Rice Cluster I. Further, we showed that liming compromised the capacity of forest and pasture soils to be a sink for methane, and instead converted formerly methane-consuming forest soils to become methane sources in only 40-80 days. Our results demonstrate that pasture management that maintains grass coverage can mitigate soil methane emissions, if compared to a bare pasture soil.

Anti-microbiota vaccines modulate the tick microbiome in a taxon-specific manner

Mateos-Hernandez, L. — 2021-05-14

Anti-tick microbiota vaccines have been shown to impact tick feeding but its specificity has not been demonstrated. In this study we aimed to investigate the impact of immune targeting of keystone microbiota bacteria on tick performance, and tick microbiota structure and function. Vaccination against Escherichia coli, the selected keystone taxon, increased tick engorgement weight and reduced bacterial diversity in Ixodes ricinus ticks compared to those that fed on mice immunized against Leuconostoc mesenteroides, a non-keystone taxon or mock-immunized group. The abundance of Escherichia-Shigella, but not Leuconostoc was significantly reduced in ticks fed on E. coli-immunized mice and this reduction was correlated with a significant increase in host antibodies (Abs) of the isotype IgM and IgG specific to E. coli proteins. This negative correlation was not observed between the abundance of Leuconostoc in ticks and anti-L. mesenteroides Abs in mice. We also demonstrated by co-occurrence network analysis, that immunization against the keystone bacterium restructure the hierarchy of the microbial community in ticks and that anti-tick microbiota vaccines reduced the resistance of networks to directed removal of taxa. Functional pathways analysis showed that immunization with a live bacterial vaccine can also induce taxon-specific changes in the abundance of pathways. Our results demonstrated that anti-tick microbiota vaccines can modulate the tick microbiome and that the modification is specific to the taxon chosen for host immunization. These results guide interventions for the control of tick infestations and pathogen infection/transmission.

Climate change and the complex dynamics of green spruce aphid--spruce plantation interactions

Thornley, J. H. M. — 2021-05-26

Aphids can have a significant impact on the growth and commercial yield of spruce plantations. Here we develop a mechanistic deterministic mathematical model for the dynamics of the green spruce aphid (Elatobium abietum Walker) growing on Sitka spruce (Picea sitchensis (Bong.) Carr.). These grow in a northern British climate in managed plantations, with planting, thinning and a 60-year rotation. Aphid infestation rarely kills the tree but can reduce growth by up to 55%. We used the Edinburgh Forest Model (efm) to simulate spruce tree growth. The aphid sub-model is described in detail in an appendix. The only environmental variable which impacts immediately on aphid dynamics is air temperature which varies diurnally and seasonally. The efm variables that are directly significant for the aphid are leaf area and phloem nitrogen and carbon. Aphid population predictions include dying out, annual, biennual and other complex patterns, including chaos. Predicted impacts on plantation yield of managed forests can be large and variable, as has been observed; they are also much affected by temperature, CO2 concentration and other climate variables. However increased CO2 concentration appears to ameliorate the severity of the effects of increasing temperatures coupled to worsening aphid infestations on plantation yield.

Long-term repair of porcine articular cartilage using cryopreservable, clinically compatible human embryonic stem cell-derived chondrocytes

Petrigliano, F. A. — 2021-05-29

Osteoarthritis (OA) impacts hundreds of millions of people worldwide, with those affected incurring significant physical and financial burdens. Injuries such as focal defects to the articular surface are a major contributing risk factor for the development of OA. Current cartilage repair strategies are moderately effective at reducing pain but often replace damaged tissue with biomechanically inferior fibrocartilage. Here we describe the development, transcriptomic ontogenetic characterization and quality assessment at the single cell level, as well as the scaled manufacturing of an allogeneic human pluripotent stem cell-derived articular chondrocyte formulation that exhibits long-term functional repair of porcine articular cartilage. These results define a new potential clinical paradigm for articular cartilage repair and mitigation of the associated risk of OA.

Mining bacterial NGS data vastly expands the complete genomes of temperate phages

Zhang, X. — 2021-07-15

Temperate phages (active prophages induced from bacteria) help control pathogenicity, modulate community structure, and maintain gut homeostasis1. Complete phage genome sequences are indispensable for understanding phage biology. Traditional plaque techniques are inapplicable to temperate phages due to the lysogenicity of these phages, which curb the identification and characterization of temperate phages. Existing in silico tools for prophage prediction usually fail to detect accurate and complete temperate phage genomes2-5. In this study, by a novel computational method mining both the integrated active prophages and their spontaneously induced forms (temperate phages), we obtained 192,326 complete temperate phage genomes from bacterial next-generation sequencing (NGS) data, hence expanded the existing number of complete temperate phage genomes by more than 100-fold. The reliability of our method was validated by wet-lab experiments. The experiments demonstrated that our method can accurately determine the complete genome sequences of the temperate phages, with exact flanking sites (attP and attB sites), outperforming other state-of-the-art prophage prediction methods. Our analysis indicates that temperate phages are likely to function in the evolution of microbes by 1) cross-infecting different bacterial host species; 2) transferring antibiotic resistance and virulence genes; and 3) interacting with hosts through restriction-modification and CRISPR/anti-CRISPR systems. This work provides a comprehensive complete temperate phage genome database and relevant information, which can serve as a valuable resource for phage research.

Serial invasions can disrupt the time course of ecosystem recovery

Karatayev, V. A. — 2021-11-01

The impacts of species invasions can subside or amplify over time as ecosystems "adapt" or additional invaders arrive. These long-term changes provide important insights into ecosystem dynamics. Yet studies of long-term dynamics are rare and often confound species impacts with coincident environmental change. We synthesize many-decade time-series across ecosystems to resolve shared changes in seven key features following invasion by quagga and zebra mussels, two widespread congeners that re-engineer and increasingly co-invade freshwaters. Six polymictic shallow lakes with long-term data sets reveal remarkably similar trends, with the strongest ecosystem impacts occurring within 5-10 years of zebra mussel invasion. Surprisingly, plankton communities then exhibited a partial, significant recovery. This recovery was absent, and impacts of initial invasion amplified, in lakes where quagga mussels outcompeted zebra mussels and more completely depleted phytoplankton. Thus, invasion impacts subside over time but can amplify with serial introductions of competing, even closely similar, taxa.

Fungal community dynamics and carbon mineralization in coarse woody debris across decay stage, tree species, and stand development stage in northern boreal forests

Hart, S. — 2022-01-31

Fungi are primary agents of coarse woody debris (CWD) decay in forests, playing an essential role in nutrient cycling and carbon storage. Characterizing fungal communities within CWD will promote further understanding of the fungal controls on CWD decomposition. We compared fungal community assemblages using alpha and beta diversity metrics, carbon mineralization, and physical and chemical properties of CWD across 3 tree species (trembling aspen [Populus tremuloides], black spruce [Picea mariana], and jack pine [Pinus banksiana]), 5 decay classes, and 2 stand development stages, differing in time-since-stand replacing disturbance (i.e., young/self-thinning and mature/steady-state) in Ontarios boreal forest region. In total, we sampled 180 individual CWD logs from 6 independent stands, with 3 replicates per each species x decay class combination at each site. We found that fungal community structure significantly differed across tree species, decay stage, and stand age. Higher proportions of white rot fungi were found in trembling aspen CWD, whereas higher proportions of brown rot fungi were found in black spruce and jack pine CWD. Proportions of specialized wood decay fungi increased with decay stage and were higher in CWD located in mature forest stands. Fungal diversity was highest in decay class 4 CWD. We found that Mn and K concentrations, total carbon, C/N ratio, carbon mineralization (mg CO2 g dry CWD-1 d-1), and moisture content were important predictors of fungal composition across CWD species and/or decay stage, though how CWD chemistry influences fungal species composition (and vice versa) is unknown. Carbon mineralization was highest in trembling aspen CWD and increased with decay stage, perhaps facilitated by increased N concentrations. This study suggests that forest management guidelines that consider both deadwood quantity and quality will support a broader range of fungal species and communities through post-disturbance stand development, thereby conserving biodiversity over the longer-term in our managed forest systems.

Deletion of proU suppresses proQ phenotypes in Escherichia coli

Smith-Frieday, M. N. — 2022-03-02

The ProQ protein interacts as an RNA chaperone with diverse RNA molecules in Escherichia coli. ProQ is implicated in the bacterial osmotic stress response. When the osmotic pressure is high, cells maintain their hydration by accumulating organic solutes denoted osmolytes. Transporters ProP and ProU (which is ProVWX) mediate osmolyte accumulation by Escherichia coli. Mutations at proQ impair ProP activity by reducing ProP levels (the ProQ transport phenotype) but do not impair ProU activity or reduce the level of ProX. The proQ- bacteria are longer than proQ+ bacteria during growth in either low or high salinity medium and they grow slowly at high salinity (the ProQ growth phenotype). In addition, spherical cells with crescent-shaped, nucleic acid-rich foci appear and cells lyse (the ProQ morphological phenotypes). In this work, the proQ transport phenotype was suppressed by deletions of proU, or by an insertion of IS5 in proU, when proP was expressed from the chromosome or from the heterologous, plasmid-based PBAD promoter. A point mutation disrupting the Walker B motif of ProV inactivated ProU but did not suppress the transport phenotype. ProP activities and ProP levels varied in parallel, so proQ and proU act at the same level to regulate ProP expression. Deletion of the proU operon also suppressed the growth and morphological phenotypes. The proU locus may overlap the gene encoding a regulatory sRNA that acts with ProQ, contributing to cellular morphogenesis and osmotic stress tolerance, or the relationship between ProQ and proU may be indirect.

A comparison of hard and soft direct methods for DNA extraction from soil

Hill, P. B. — 2022-03-08

Nucleic acid extraction is the first step in molecular biology studies of soil bacterial communities. The most common used soil DNA extraction method is the direct, hard extraction Mobio method, which uses bead beating to lyse bacteria. In this study we compared the Mobio method with a soft, enzymatic lysis extraction method. Next generation sequencing (Illumina and Pyrosequencing) of amplicons generated from four 16S primer pairs and DNA from 12 soils and 3 composts was used to compare the two extraction methods. Four bacterial orders, the delta proteobacterial Desulfuromonadales and gamma proteobacterial Pseudomonadales, Enterobacteriales, and Alteromonadales were more common in amplicons from soft extracted DNA, sometimes by two orders of magnitude. These groups can be a significant fraction of the bacterial population. For example the Pseudomonadales made up to 16 % and Enterobacteriales 10% of amplicons from Soft extracted DNA. The JG30-KF-CM45 order was under extracted by the enzymatic lysis extraction method. Results differed more by primer choice than extraction method and the phylogenetic resolution of differences between extraction methods changed with primer choice. Given how often Mobio extraction is used, these proteobacterial orders are probably under-represented in the studies of soil bacteria that use nucleic acid methods. Further improvements in soil DNA extraction are needed. Amplicons sequencing studies should use a range of different primers to confirm the phylogenetic resolution of their results. ImportanceSeveral large scale studies of soil bacteria that compare thousands of soil samples across continents have used the Mobio method for DNA extraction. Large scale studies like these are increasing with the recent establishment of the Global Soil Biodiversity Observation Network (Soil BON), which also uses the Mobio method. The results of this work will be used to make policy decisions about how to manage the soil and may be a guide for bioprospectors. As the Mobio method is so widely used, it is important to know its limitations. Studies that use the Mobio method underestimate the fraction of several proteobacterial groups. Most notably the Enterobacteria and Pseudomonas can be under extracted by 10-100 fold. The degree of under extraction varies with different soils.

A free classified advertising website is a high-risk unregulated source of domestic wild boar, pot-bellied pigs, and their hybrids with domestic swine in Canada: Implications for African Swine Fever Risk Preparedness

MacDonald, A. M. — 2022-03-16

Invasive wild pigs are a global problem and are established in large areas across Canada. Domestic European wild boar were first introduced to Canadian farms in the 1980s, and their escape and intentional release have since led to free-ranging populations, which have expanded rapidly in numbers and range. Wild pigs are associated with widespread damage to crops and natural environments, as well as risks of disease transmission. African Swine Fever (ASF) is a highly contagious viral disease of global concern, that can spread between domestic and free-ranging swine and cause catastrophic economic impacts. As ASF preparedness requires a detailed understanding of the distribution and movements of all pig types, this paper aimed to characterize an unregulated grey swine market in Canada to understand risks to domestic swine production and potential contributions to free-ranging wild pig populations. Kijiji.ca is a free Canadian internet classified advertising service, almost exclusively cash-based with no receipts and few, if any, records kept of transactions. We monitored Kijiji for sales of domestic wild boar, pot-bellied pigs, and their hybrids across Canada over two months from April 28 - June 30, 2021. Data was collected from all advertisements, including how the seller labelled pigs breed, age, sex, number for sale, sexual intactness, presence of tattoos and/or ear tags, as well as the date and location of the posting. Locations were mapped and compared to the spatial distribution of existing free-ranging wild pigs in Canada to identify areas they were likely to facilitate new populations or potentially supplement and genetically diversify existing populations. We identified 151 advertisements on Kijiji, the most coming from Ontario (34.4%; n = 52) and Alberta (29.1%; n = 44), followed by Saskatchewan (11.3%; n = 17), British Columbia (10.6%; n = 16), Nova Scotia (7.3%; n = 11), New Brunswick (4.0%; n = 6), Manitoba (2.0%; n = 3), Quebec (0.7%; n = 1), and Prince Edward Island (0.7%; n = 1). No listings were observed in Newfoundland and Labrador, or Yukon, Nunavut and Northwest Territories. Overall, 62 (41%) of advertisements were within watersheds with existing wild pigs that may contribute to supplementing additional swine, and 89 (59%) were within watersheds where wild pigs have not yet been identified. African swine fever preparedness should include policies and action to address this unmonitored and unregulated sale of swine in Canada, including ear tags, tattoos, genetic analysis, and mandatory reporting.

Population genomics provide insights into the global genetic structure of Colletotrichum graminicola, the causal agent of maize anthracnose

Rogerio, F. — 2022-05-09

BackgroundColletotrichum graminicola, the causal agent of maize anthracnose, is an important crop disease worldwide. Understanding the genetic diversity and mechanisms underlying genetic variation in pathogen populations is crucial to the development of effective control strategies. The genus Colletotrichum is largely recognized as asexual, but several species have been reported to have a sexual cycle. Here, we employed a population genomics approach to investigate the genetic diversity and reproductive biology of C. graminicola isolates infecting maize. We sequenced 108 isolates of C. graminicola collected in 14 countries using restriction site-associated DNA sequencing (RAD-Seq) and whole-genome sequencing (WGS). ResultsClustering analyses based on single-nucleotide polymorphisms showed populational differentiation at a global scale, with three genetic groups delimited by continental origin, compatible with short-dispersal of the pathogen, and geographic subdivision. Distinct levels of genetic diversity were observed between these clades, suggesting different evolutionary histories. Intra and inter-continental migration was predicted between Europe and South America, likely associated with the movement of contaminated germplasm. Low clonality and evidence of genetic recombination were detected from the analysis of linkage disequilibrium and the pairwise homoplasy index (PHI) test for clonality. We show evidence that even if rare (possibly due to losses of sex and meiosis-associated genes) C. graminicola can undergo sexual recombination based on lab assays and genomic analyses. ConclusionsOur results support hypotheses of intra and intercontinental pathogen migration and genetic recombination with great impact on C. graminicola population structure.

Thirty years of deforestation within the entire ranges of nine endangered lemur species (3 CR, 4 EN, 2 VU) in northwestern Madagascar

Schuessler, D. — 2022-05-24

Forest cover change is of particular concern in tropical regions. In this study, we investigate the degree of deforestation in the entire ranges of nine highly threatened lemur species in northwestern Madagascar. Landsat satellite images were acquired from four different time stages (1990, 2000, 2011, 2020), classified into forest/non-forest, and changes quantified. Forest cover declined from 17.5% to 9.3% within the last 30 years. This decline varied across four protected areas (PAs) investigated: the forest cover of Ankarafantsika National Park (ANP) declined only moderately over time (from 76.3% to 67.4%), while it declined drastically in other PAs (e.g., from 54.9% to 18.9%, Bongolava Forest Corridor). Two lemur taxa are most affected (Lepilemur otto, Microcebus bongolavensis) by having only very few isolated forest patches left within their ranges (approximately 542.7 km{superscript 2}). For two other species (L. ahmansoni, L. aeeclis), most of the remaining forest is concentrated in two coastal PAs (in total 627.2 and 477.9 km{superscript 2}, respectively), while those species occurring inside ANP (5 taxa) experienced rather stable forest coverage until 2020. A reversal of these deforestation trends and active reforestation measures are desperately needed to reduce habitat loss for these nine lemur species. A practical experience-based guideline is therefore provided.

Temperature dependence of competitive ability differs from that of growth rate

Sunday, J. M. — 2022-07-22

The effect of climate warming on future community composition is expected to be contingent on competitive outcomes, yet we currently lack mechanistic ecological understanding of how temperature affects competitive ability. Here, we combine resource competition theory with metabolic scaling theory and test hypotheses about how the temperature dependence of competitive ability changes with temperature. We find that the minimum resource requirement for growth, R* - an inverse indicator of competitive ability in phytoplankton - changes with temperature following a U-shaped pattern in all four species tested. The shape of temperature-dependence of competitive ability is systematically different from the temperature-dependence of population growth rates, both in our experiments and in collated data from previous studies. Our results suggest that exploitative competitive success is highest at temperatures that are sub-optimal for growth, and declines rapidly at both cold and warm ends of the thermal performance curve.

Environmental, social, and morphological drivers of fission-fusion dynamics in a social ungulate

Le Goff, M. — 2022-09-23

Social groups exist because individuals within the group accrue a net benefit from sharing space. The profitability of sociality, however, varies with ecological context. As ecological context varies, tension emerges among the costs and benefits of social grouping. Fission-fusion societies are fluid in their group dynamics across spatial and temporal contexts, permitting insights into how context affects whether animals choose to join or depart a group. We tested four non-mutually exclusive hypotheses driving variation in fission and fusion in caribou: the risky places, environment heterogeneity, activity budget, and social familiarity hypotheses. The risky places hypothesis predicts animals are unlikely to diffuse when habitats are open and risk of predation is elevated. The habitat heterogeneity hypothesis predicts that fission is more likely in a heterogeneous landscape due to the rising conflicts of interest between group members. The activity budget hypothesis predicts dyads associate by body size due to similar food passage rates. The social cohesion hypothesis predicts that familiar individuals are less likely to fission. We tested the hypotheses using time-to-event (time before fission) analyses and a linear model that assesses spatial, social, and body size relationships among female caribou (n = 22) on Fogo Island, Newfoundland, Canada. Contrary to our prediction for risky places, probability of fission was not influenced by habitat openness. The hypothesis of environmental heterogeneity was partially supported, as caribou remained less cohesive in environments with a higher richness of habitats. No direct evidence emerged to support the activity budget hypothesis. However, it appears that caribou maintain the strongest social bonds among variably sized individuals and these social bonds do decrease the propensity to split. Collectively, our findings showed that social interactions may depend not only on individual identity and characteristics, but also the spatial context in which these interactions occur.

Acclimating to degraded environments: The social rationale for swift action on restoration

Karatayev, V. A. — 2022-09-28

As environmental degradation progresses, economies and societies adapt to the loss of ecosystem services and public attention to degradation subsides. In systems experiencing such societal acclimation to degradation, net incentives for stakeholder mitigation peak during early degradation phases and subside over time. Using harmful algae blooms in western Lake Erie as a case study, we illustrate how declines in public attention and societal reliance on lake recreation (i.e., finding recreation alternatives) reduce the incentives for stakeholders to reduce pollution runoff (i.e., mitigation efforts throughout the watershed). We then analyze how acclimation can affect a broad array of conservation challenges by developing a general socio-ecological model of societal response to degradation. We find that delays in initiating stakeholder-driven mitigation efforts can exponentially prolong restoration projects. Furthermore, when alleviating intense degradation relies upon voluntary commitments by many individuals, windows of opportunity for mitigation can be very limited because feedback loops of societal adaptation doom late restoration efforts to failure and lock human-environment systems into degraded states. These windows of opportunity can be particularly narrow when a) stakeholder mitigation requires supportive public opinion or b) even modestly valuable alternative services are available in degraded ecosystems. In such cases, maintaining undegraded human-environment regimes may hinge on quickly initiating stakeholder mitigation movements and allocating limited government conservation funds soon after degradation begins instead of spreading mitigation efforts out over decades. Such initiatives, regardless of whether acclimation is slow or rapid in a given system, also greatly accelerate the pace of environmental restoration. Significance StatementAs societies acclimate to degraded environments, mitigation efforts that hinge on action by many stakeholders can erode. Developing a socio-ecological model of acclimation, we reveal how social and environmental processes intertwine to create alternative stable socio-ecological regimes, with either: 1) undegraded ecosystem states sustained by widespread mitigation adoption, or 2) degraded states where societies neither maintain nor continue relying on traditional, local ecosystem services. This dynamic places a premium on prompt mitigation efforts, which may face narrow opportunity windows to get started and avert degraded regimes in systems that rely on stakeholder-driven mitigation. Moreover, in any system requiring stakeholder action, societal acclimation will increase the importance of early action because decaying mitigation incentives exponentially lengthen restoration efforts.

From Pigs to Silkworms: Cognition and Welfare across 10 Farmed Taxa

Miller, R. — 2022-11-14

Billions of animals across many taxa are extensively farmed, with critical impacts on animal welfare. Societal efforts to reduce animal suffering lack rigorous and systematic approaches that facilitate maximising welfare improvements, such as informed funding allocation decisions. We present a multi-measure, cross-taxa framework for modelling differences in pain, suffering, and related cognition to assess whether certain animals have larger welfare ranges (how well or badly animals can fare). Measures include behavioural flexibility, cognitive sophistication, and general learning. We evaluated 90 empirically detectable proxies for cognition and welfare range (henceforth proxies) in pigs, chickens, carp, salmon, octopus, shrimp, crabs, crayfish, bees, and silkworms. We grouped a subset of proxies into: A) 10 ideal proxies and B) 10 less ideal proxies but with sufficient data for interspecies comparisons. We graded the strength of evidence per proxy across taxa, and constructed a cognition and welfare range profile, with overall judgement scores (ranging from likely no/low confidence to yes/very high confidence). We discuss the implications of comparisons and highlight key avenues for future research. This work is timely, given recent indications of significant political will towards reducing animal suffering, such as the inclusion of cephalopods and decapods in the Animal Welfare (Sentience) Bill following a UK government-commissioned research review. Given the novelty and robustness of our review, we believe it sets a new standard for investigating interspecies comparisons of cognition and welfare ranges and helps inform future research. This should help streamline funding allocations and improve the welfare of millions of farmed animals. Graphical/ Visual Abstract and Caption O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Cognition and welfare in farmed animals - from pigs to silkworms (Free stock images: http://www.pixabay.com) C_FIG_DISPLAY

Recording animal-view videos of the natural world

Vasas, V. — 2022-11-23

Plants, animals, and fungi display a rich tapestry of colors. Animals, in particular, use colors in dynamic displays performed in spatially complex environments. In such natural settings, light is reflected or refracted from objects with complex shapes that cast shadows and generate highlights. In addition, the illuminating light changes continuously as viewers and targets move through heterogeneous, continually fluctuating, light conditions. Although traditional spectrophotometric approaches for studying colors are objective and repeatable, they fail to document this complexity. Worse, they miss the temporal variation of color signals entirely. Here, we introduce hardware and software that provide ecologists and filmmakers the ability to accurately record animal-perceived colors in motion. Specifically, our Python codes transform photos or videos into perceivable units (quantum catches) for any animal of known photoreceptor sensitivity. We provide the plans, codes, and validation tests necessary for end-users to capture animal-view videos. This approach will allow ecologists to investigate how animals use colors in dynamic behavioral displays, the ways natural illumination alters perceived colors, and other questions that remained unaddressed until now due to a lack of suitable tools. Finally, our pipeline provides scientists and filmmakers with a new, empirically grounded approach for depicting the perceptual worlds of non-human animals.

Within-species variation in the gut microbiome of fish is driven by the interaction of light intensity and genetic background

Evangelista, C. — 2023-02-17

Unravelling evolution-by-environment interactions on the gut microbiome is particularly relevant considering the unprecedented level of human-driven disruption of the ecological and evolutionary trajectories of species. Here, we aimed to evaluate whether an evolutionary response to size-selective mortality influences the gut microbiome of medaka (Oryzias latipes), how environmental conditions interact with the genetic background of medaka on their microbiota, and the association between microbiome diversity and medaka growth-related traits. To do so, we studied two lineages of medaka with known divergence in foraging efficiency and life history raised under antagonistic size-selective regimes for 10 generations (i.e. the largest or the smallest breeders were removed to mimic fishing-like or natural mortality). In pond mesocosms, the two lineages were subjected to contrasting population density and light intensity (used as proxies of resource availability). We observed significant differences in the gut microbiome composition and richness between the two lines, and this effect was mediated by light intensity. The bacterial richness of fishing-like medaka (small-breeder line) was reduced by 34% under low-light conditions compared to high-light conditions, while it remained unchanged in natural mortality-selected medaka (large-breeder line). However, the observed changes in bacterial richness did not correlate with changes in adult growth rate or body condition. Given the growing evidence about the gut microbiomes importance to host health, more in-depth studies are required to fully understand the role of the microbiome in size-selected organisms and the possible ecosystem-level consequences.

Genomic, proteomic, and phylogenetic analysis of spounaviruses indicates paraphyly of the order Caudovirales

Barylski, J. — 2017-11-16

It is almost a cliche that tailed bacteriophages of the order Caudovirales are the most abundant and diverse viruses in the world. Yet, their taxonomy still consists of a single order with just three families: Myoviridae, Siphoviridae, and Podoviridae. Thousands of newly discovered phage genomes have recently challenged this morphology-based classification, revealing that tailed bacteriophages are genomically even more diverse than once thought. Here, we evaluate a range of methods for bacteriophage taxonomy by using a particularly challenging group as an example, the Bacillus phage SPO1-related viruses of the myovirid subfamily Spounavirinae. Exhaustive phylogenetic and phylogenomic analyses indicate that the spounavirins are consistent with the taxonomic rank of family and should be divided into at least five subfamilies. This work is a case study for virus genomic taxonomy and the first step in an impending massive reorganization of the tailed bacteriophage taxonomy.

MethylSight: Taking a wider view of lysine methylation through computer-aided discovery to provide insight into the human methyl-lysine proteome

Biggar, K. K. — 2018-03-02

Protein Lys methylation plays a critical role in numerous cellular processes, yet it has been challenging to identify Lys methylation in a systematic manner. We present here an approach combining in silico prediction with targeted mass spectrometry (MS) to identify Lys methylation (Kme) sites at the proteome level. We have developed MethylSight, a program that predicts Kme events solely on physicochemical and biochemical properties of putative methylation sites, which can then be validated by targeted MS. Using this approach, we have identified 70 new histone Kme marks with a 90% validation rate. H2BK43me2, which undergoes dynamic changes during stem cell differentiation, is found to be a substrate of KDM5b. Furthermore, MethylSight predicts ~50,000 Kme sites in non-histone proteins with high confidence, suggesting that Lys methylation is a prevalent post-translational modification. Our work provides a useful resource for systematic exploration of the role of Lys methylation in human health and disease.

Illuminating the microbiome’s dark matter: a functional genomic toolkit for the study of human gut Actinobacteria

Bisanz, J. E. — 2018-05-01

Despite the remarkable evolutionary and metabolic diversity found within the human microbiome, the vast majority of mechanistic studies focus on two phyla: the Bacteroidetes and the Proteobacteria. Generalizable tools for studying the other phyla are urgently needed in order to transition microbiome research from a descriptive to a mechanistic discipline. Here, we focus on the Coriobacteriia class within the Actinobacteria phylum, detected in the distal gut of 90% of adult individuals around the world, which have been associated with both chronic and infectious disease, and play a key role in the metabolism of pharmaceutical, dietary, and endogenous compounds. We established, sequenced, and annotated a strain collection spanning 14 genera, 8 decades, and 3 continents, with a focus on Eggerthella lenta. Genome-wide alignments revealed inconsistencies in the taxonomy of the Coriobacteriia for which amendments have been proposed. Re-sequencing of the E. lenta type strain from multiple culture collections and our laboratory stock allowed us to identify errors in the finished genome and to identify point mutations associated with antibiotic resistance. Analysis of 24 E. lenta genomes revealed an "open" pan-genome suggesting we still have not fully sampled the genetic and metabolic diversity within this bacterial species. Consistent with the requirement for arginine during in vitro growth, the core E. lenta genome included the arginine dihydrolase pathway. Surprisingly, glycolysis and the citric acid cycle was also conserved in E. lenta despite the lack of evidence for carbohydrate utilization. We identified a species-specific marker gene and validated a multiplexed quantitative PCR assay for simultaneous detection of E. lenta and specific genes of interest from stool samples. Finally, we demonstrated the utility of comparative genomics for linking variable genes to strain-specific phenotypes, including antibiotic resistance and drug metabolism. To facilitate the continued functional genomic analysis of the Coriobacteriia, we have deposited the full collection of strains in DSMZ and have written a general software tool (ElenMatchR) that can be readily applied to novel phenotypic traits of interest. Together, these tools provide a first step towards a molecular understanding of the many neglected but clinically-relevant members of the human gut microbiome.

Experimental increases in glucocorticoids alter function of the neuroendocrine stress axis in wild red squirrels without negatively impacting survival and reproduction

van Kesteren, F. — 2018-04-26

Hormones such as glucocorticoids (colloquially referred to as "stress hormones") have important effects on animal behavior and life history traits, yet most of this understanding has come through correlative studies. While experimental studies offer the ability to assign causality, there are important methodological concerns that are often not considered when manipulating hormones, including glucocorticoids, in wild animals. In this study, we examined how experimental elevations of cortisol concentrations in wild North American red squirrels (Tamiasciurus hudsonicus) affected their hypothalamic-pituitary-adrenal (HPA) axis reactivity, and life history traits including body mass, litter survival, and adult survival. The effects of exogenous cortisol on plasma cortisol concentrations depended on the time between treatment consumption and blood sampling. In the first nine hours after consumption of exogenous cortisol, individuals had significantly higher true baseline plasma cortisol concentrations, but adrenal gland function was impaired as indicated by their dampened response to capture and handling and to injections of adrenocorticotropic hormone compared to controls. Approximately 24 hours after consumption of exogenous cortisol, individuals had much lower plasma cortisol concentrations than controls, but adrenal function was restored. Corticosteroid binding globulin (CBG) concentrations were also significantly reduced in squirrels treated with cortisol. Despite these profound shifts in the functionality of the HPA axis, squirrel body mass, offspring survival, and adult survival were unaffected by experimental increases in cortisol concentrations. Our results highlight that even short-term experimental increases in glucocorticoids can affect adrenal gland functioning and CBG concentrations but without other side-effects.

Indirect effects of territorial neighbors on the timing of spring breeding may counteract changes in selection in North American red squirrels

Fisher, D. N. — 2018-05-23

Organisms can affect one anothers phenotypes when they socially interact. Indirect genetic effects occur when an individuals phenotype is affected by genes expressed in another individual. These heritable effects can enhance or reduce adaptive potential, thereby accelerating or reversing evolutionary change. Quantifying these social effects is therefore crucial for our understanding of evolution, yet estimates of indirect genetic effects in wild animals are limited to dyadic interactions. We estimated indirect phenotypic and genetic effects, and their covariance with direct effects, for the date of spring breeding in North American red squirrels (Tamiasciurus hudsonicus) living in an array of territories of varying spatial proximity. Additionally, we estimated variance parameters and the strength of selection at low and high population densities. Social effects of neighbours on the date of spring breeding were weak at low, but stronger at high population densities. Indirect phenotypic effects accounted for a larger amount of variation in the date of breeding than direct differences among-individuals, although the genetic component to these indirect effects was not statistically significant. Nevertheless, the estimated effect size was large enough to suggest that indirect genetic effects could alter evolutionary change, resulting in less change at high densities despite stronger selection. Despite the difficulty in estimating them precisely, indirect genetic effects have clear potential to alter evolutionary trajectories in any natural systems where organisms interact.

Direct immunosensing of avian influenza A virus in whole blood using hybrid nanocomposites

Buozis, J. — 2018-06-11

A sandwich-based electrochemical immunosensor was designed for detection of avian influenza virus (AIV) strains H5N1 and H4N6. This sensor was developed using gold-graphene nanocomposites, immobilized viral antibodies, and CdTe quantum dot electrochemical tagging. The nanocomposites were formed by the simultaneous reduction of a gold salt and graphene using hydroquinone as the reducing agent, thus producing non-spherical gold nanoparticles on graphene sheets. Viral antibodies were immobilized on nanocomposites and CdTe quantum dots through N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysuccinimide chemistry. Cyclic voltammetry studies were used to validate the detection of H5N1 surface protein and H4N6 inactivated virus. The immunosensor detected H5 protein in phosphate buffer solution (pH 7.4) with a limit of detection (LOD) of 10 fg/mL and a linear detection range was established for 10 ng/mL to 10 pg/mL. The biosensor detected H4N6 in three parts diluted whole chicken blood with a LOD of 1.28x10-7 hemagglutinating units (HAU). Commercial ELISA testing for H5N1 and H4N6 showed limits of detection of 10 ng/mL and 0.128 HAU, respectively. The sensor showed 106-fold increased detection of H4N6 virus in blood in comparison to its commercial ELISA kit counterpart. The developed immunosensor effectively change the way avian influenza is detected, monitored, and controlled; transforming time-consuming reactive methods, into rapid predictive technology.

Objective Evaluation of Multiple Sclerosis Lesion Segmentation using a Data Management and Processing Infrastructure

Commowick, O. — 2018-07-13

We present a study of multiple sclerosis segmentation algorithms conducted at the international MICCAI 2016 challenge. This challenge was operated using a new open-science computing infrastructure. This allowed for the automatic and independent evaluation of a large range of algorithms in a fair and completely automatic manner. This computing infrastructure was used to evaluate thirteen methods of MS lesions segmentation, exploring a broad range of state-of-the-art algorithms, against a high-quality database of 53 MS cases coming from four centers following a common definition of the acquisition protocol. Each case was annotated manually by an unprecedented number of seven different experts. Results of the challenge highlighted that automatic algorithms, including the recent machine learning methods (random forests, deep learning, ...), are still trailing human expertise on both detection and delineation criteria. In addition, we demonstrate that computing a statistically robust consensus of the algorithms performs closer to human expertise on one score (segmentation) although still trailing on detection scores.

Revealing the Complexities of Metabarcoding with a Diverse Arthropod Mock Community

Braukmann, T. W. — 2018-10-02

DNA metabarcoding is an attractive approach for monitoring biodiversity. However, it is subject to biases that often impede detection of all species in a sample. In particular, the proportion of sequences recovered from each species depends on its biomass, mitome copy number, and primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 BINs, a species proxy. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body partitions (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR-amplified separately (Single Leg) and then pooled. The amplicon generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, other variables did. As expected, the best recovery was obtained from the Single Leg treatment, but the Bulk Abdomen produced a more uniform read abundance than the Bulk Leg or Composite Leg samples. Primer choice also influenced species recovery. Our results reveal how variation in protocols can have substantive impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote. Although metabarcoding is a powerful approach, further optimization of analytical protocols is crucial to obtain reproducible results and increase its cost-effectiveness.

High-fat diet intake modulates maternal intestinal adaptations to pregnancy, and results in placental hypoxia and impaired fetal gut development

Gohir, W. — 2018-10-05

Shifts in maternal intestinal microbiota have been implicated in metabolic adaptations to pregnancy. In this study we investigated how high-fat diet intake impacts the maternal gut microbiota, intestinal inflammation and gut barrier integrity, placental inflammation, and fetal intestinal development at E18.5. High-fat diet (HFD) was associated with decreased relative abundancesof SCFA producing genera during pregnancy. These diet-induced shifts paralleled decreased maternal intestinal mRNA levels of SCFA receptor Gpr41, modestly decreased cecal butyrate, and altered mRNA levels of inflammatory cytokines and immune cell markers in the maternal intestine. Maternal HFD resulted inimpaired gut barrier integrity, with corresponding increases in circulating maternal levels of LPS and TNF.Placentafromhigh-fat fed damsdemonstrated blood vessel immatu-rityand hypoxia, decreased freecarnitine, acylcarnitine derivatives, TMAO, as well as altered mRNA levels of inflammation, autophagy and ER stress markers. HFD exposed fetuses had increased activation of NF-{kappa}B and inhibition of the unfolded protein response in the developing intestine. Together, these data suggest that high-fat diet intake prior to and during pregnancy shifts the composition of the maternal gut microbiota and impairs gut barrier integrity, resulting in increased maternal circulating LPS, which may ultimate contribute to changes in placental vasculariza-tion and fetal gut development.nnFunding informationFarncombe Family Digestive Health Research Institute (KMK); Canadian Institute of Health Research (CJB); Canada Research Chairs Program (MGS, DMS); Natural Sciences and Engineering Research Council of Canada, Genome Canada (PBM).

Indirect effects on fitness between individuals that have never met via an extended phenotype

Fisher, D. — 2018-10-13

Interactions between organisms are ubiquitous and have important consequences for phenotypes and fitness. Individuals can even influence those they never meet, if they have extended phenotypes which mean the environments others experience are altered. North American red squirrels (Tamiasciurus hudsonicus) guard food hoards, an extended phenotype that typically outlives the individual and is almost always inherited by non relatives. Hoarding by previous owners can therefore influence subsequent owners. We found that red squirrels bred earlier and had higher lifetime fitness if the previous owner was a male. This was driven by hoarding behaviour, as males and mid-aged squirrels had the largest hoards, and these effects persisted across owners, such that if the previous owner was male or died in mid-age subsequent occupants had larger hoards. Individuals can, therefore, influence each others resource dependent traits and fitness without meeting via extended phenotypes, and so the past can influence contemporary population dynamics.

Individual variation in the dear enemy phenomenon via territorial vocalizations in red squirrels

Robertson, J. G. — 2018-10-17

Territoriality arises when the benefits of resources exceed the costs of defending them. The dear enemy phenomenon, where familiar territorial neighbours refrain from intruding on one another and mutually reduce their defensive efforts, allows for reduction of these costs but requires discrimination between conspecifics. We hypothesized that territorial vocalizations in red squirrels (Tamiasciurus hudsonicus) are used for this discrimination. We performed a speaker replacement experiment where red squirrels (n = 41) were temporarily removed from their territories and replaced with a speaker broadcasting their own call, an unfamiliar call, or silence. Contrary to our prediction, there were no differences in overall intrusion risk among our three playbacks, but the identity of intruders did vary. Existing variation in familiarity within territorial neighbourhoods should be considered, rather than the binary classification of familiar or stranger, when studying dear enemy effects. We also discuss the variable importance of silence in acoustic territorial populations.

Red squirrel territorial vocalizations deter intrusions by conspecific rivals

Siracusa, E. — 2018-10-22

In many species, territory defense is thought to be one of the primary functions of acoustic communication. North American red squirrels are a territorial species in which rattles have long been thought to be the principal signal communicating territory ownership. These vocalizations have been assumed to deter intruders, thus reducing energy costs and the risk of injury associated with direct aggressive interactions. However, this hypothesis has not been directly tested. Here we used a speaker occupation experiment to test whether red squirrel rattles function to deter conspecific rivals. We studied 29 male squirrels and removed each individual from his territory twice in a paired design. During the experimental treatment we simulated the owners presence after its removal by broadcasting the owners rattle from a loudspeaker at the center of the territory once every seven minutes. During the control treatment the territory was left in silence after the temporary removal of the owner. We found that the presence of a speaker replacement reduced the probability of intrusion by 34% and increased the latency to first intrusion by 7%, providing support for the hypothesis that rattles play an active role in reducing intrusion risk. However, intrusions were not completely averted by the speaker replacement, indicating that vocalizations alone are not sufficient without other cues of the territory owner.

Red squirrels mitigate costs of territory defence through social plasticity

Siracusa, E. — 2018-10-26

For territorial species, the ability to be behaviourally plastic in response to changes in their social environment may be beneficial by allowing individuals to mitigate conflict with conspecifics and reduce the costs of territoriality. Here we investigated whether North American red squirrels (Tamiasciurus hudsonicus) were able to minimize costs of territory defence by adjusting behaviour in response to the familiarity of neighbouring conspecifics. Since red squirrels living in familiar neighbourhoods face reduced intrusion risk, we predicted that increasing familiarity among territorial neighbours would allow squirrels to spend less time on territorial defence and more time in the nest. Long-term behavioural data (1995-2004) collected from the same squirrels across several different social environments indicated that red squirrels reduced rates of territorial vocalizations and increased nest use in response to increasing familiarity with neighbours. In contrast, cross-sectional data (2015-2016), which provided observations from each individual in a single social environment, did not provide evidence of this plasticity. Post-hoc analyses revealed that evidence of social plasticity in this system was primarily due to within-individual changes in behaviour, which we were unable to estimate in the cross-sectional data. Our results demonstrate that red squirrels can reduce the costs of territoriality by appropriately adjusting behaviour in response to changes in their social environment. However, our results also suggest that estimating plasticity by comparing behaviour among individuals (i.e. cross-sectional analyses) may not always be reliable. Our ability to detect these effects may therefore depend on having data with multiple observations from the same individuals across different social environments.

Stress activity is not predictive of coping style in North American red squirrels

Westrick, S. E. — 2018-11-07

Individuals vary in their behavioral and physiological responses to environmental changes. These behavioral responses are often described as coping styles along a proactive-reactive continuum. Studies in laboratory populations often, but not always, find that behavioral responses and physiological responses to stressors covary, where more proactive (more aggressive and active) individuals have a lower physiological stress response, specifically as measured by hypothalamic-pituitary-adrenal (HPA) axis activity. These studies support the possibility of hormonal pleiotropy underlying the presentation of behaviors that make up the proactive-reactive phenotype. However, recent research in wild populations is equivocal, with some studies reporting the same pattern as found in many controlled laboratory studies, whereas others do not. We tested the hypothesis that physiological and behavioral stress responses are correlated in wild adult North American red squirrels (Tamiasciurus hudsonicus). We used fecal cortisol metabolites (FCMs) as a non-invasive, integrated estimate of circulating glucocorticoids for our measurement of HPA axis activity. We found that FCM concentrations were not correlated with three measures of behavioral coping styles (activity, aggression, and docility) among individuals. This does not support the hypothesis that hormonal pleiotropy underlies a proactive-reactive continuum of coping styles. Instead, our results support the "two-tier" hypothesis that behavioral and physiological stress responses are independent and uncorrelated traits among individuals in wild populations that experience naturally varying environments rather than controlled environments. If also found in other studies, this may alter our predictions about the evolutionary consequences of behavioral and endocrine coping styles in free-living animals.nnSignificance StatementIndividuals vary in how they respond to stressors through behavior and physiology, but we find the two responses are independent in wild animals. Many laboratory studies find links between the behavioral and physiological stress responses, however studies conducted with wild populations are less conclusive. In wild North American red squirrels, independence between the physiological response and behavioral response may allow adaptive responses to a changing environment without pleiotropic constraint.

Multiple modes of convergent adaptation in the spread of glyphosate-resistant Amaranthus tuberculatus

Kreiner, J. M. — 2018-12-17

The selection pressure exerted by herbicides has led to the repeated evolution of herbicide resistance in weeds. The evolution of herbicide resistance on contemporary timescales in turn provides an outstanding opportunity to investigate key questions about the genetics of adaptation, in particular, the relative importance of adaptation from new mutations, standing genetic variation, or geographic spread of adaptive alleles through gene flow. Glyphosate-resistant Amaranthus tuberculatus poses one of the most significant threats to crop yields in the midwestern United States (1), with both agricultural populations and herbicide resistance only recently emerging in Canada (2, 3). To understand the evolutionary mechanisms driving the spread of resistance, we sequenced and assembled the A. tuberculatus genome and investigated the origins and population genomics of 163 resequenced glyphosate-resistant and susceptible individuals from Canada and the USA. In Canada, we discovered multiple modes of convergent evolution: in one locality, resistance appears to have evolved through introductions of preadapted US genotypes, while in another, there is evidence for the independent evolution of resistance on genomic backgrounds that are historically non-agricultural. Moreover, resistance on these local, non-agricultural backgrounds appears to have occurred predominantly through the partial sweep of a single haplotype. In contrast, resistant haplotypes arising from the midwestern US show multiple amplification haplotypes segregating both between and within populations. Therefore, while the remarkable species-wide diversity of A. tuberculatus has facilitated geographic parallel adaptation of glyphosate resistance, more recently established agricultural populations are limited to adaptation in a more mutation-limited framework.nnSignificanceWhile evolution is often thought of as playing out over millions of years, adaptation to new enviroments can occur in real time, presenting key opportunities to understand evolutionary processes. An important example comes from agriculture, where many weeds have evolved herbicide resistance. We have studied glyphosate resistant Amaranthus tuberculatus, a significant threat to crop yields in the midwestern US and Canada. Genome analyses showed that rapid evolution can either occur by "borrowing" resistance alleles from other locations, or by de novo evolution of herbicide resistance in a genetic background that was not previously associated with agriculture. Differences in recent evolutionary histories have thus favored either adaptation from pre-existing variation or new mutation in different parts of the A. tuberculatus range.

Comprehensive biodiversity analysis via ultra-deep patterned flow cell technology: a case study of eDNA metabarcoding seawater

Singer, G. — 2019-01-09

1The characterization of biodiversity is a crucial element of ecological investigations as well as environmental assessment and monitoring activities. Increasingly, amplicon-based environmental DNA metabarcoding (alternatively, marker gene metagenomics) is used for such studies given its ability to provide biodiversity data from various groups of organisms simply from analysis of bulk environmental samples such as water, soil or sediments. The Illumina MiSeq is currently the most popular tool for carrying out this work, but we set out to determine whether typical studies were reading enough DNA to detect rare organisms (i.e., those that may be of greatest interest such as endangered or invasive species) present in the environment. We collected sea water samples along two transects in Conception Bay, Newfoundland and analyzed them on the MiSeq with a sequencing depth of 100,000 reads per sample (exceeding the 60,000 per sample that is typical of similar studies). We then analyzed these same samples on Illuminas newest high-capacity platform, the NovaSeq, at a depth of 7 million reads per sample. Not surprisingly, the NovaSeq detected many more taxa than the MiSeq thanks to its much greater sequencing depth. However, contrary to our expectations this pattern was true even in depth-for-depth comparisons. In other words, the NovaSeq can detect more DNA sequence diversity within samples than the MiSeq, even at the exact same sequencing depth. Even when samples were reanalyzed on the MiSeq with a sequencing depth of 1 million reads each, the MiSeqs ability to detect new sequences plateaued while the NovaSeq continued to detect new sequence variants. These results have important biological implications. The NovaSeq found 40% more metazoan families in this environment than the MiSeq, including some of interest such as marine mammals and bony fish so the real-world implications of these findings are significant. These results are most likely associated to the advances incorporated in NovaSeq especially patterned flow cell, which prevents similar sequences that are neighbours on the flow cell (common in metabarcoding studies) from being erroneously merged into single spots by the sequencing instrument. This study sets the stage for incorporating eDNA metabarcoding in comprehensive analysis of oceanic samples in a wide range of ecological and environmental investigations.

Phenotypic evolution is more constrained in simpler food webs

Barbour, M. A. — 2019-01-23

Global change is simplifying the structure of ecological networks; however, we are currently in a poor position to predict how these simplified communities will affect the evolutionary potential of remaining populations. Theory on adaptive landscapes provides a framework for predicting how selection constrains phenotypic evolution, but often treats the community context of evolving populations as a "black box". Here, we integrate ecological networks and adaptive landscapes to examine how changes in food-web complexity shape evolutionary constraints. We conducted a field experiment that manipulated the diversity of insect parasitoids (food-web complexity) that were able to impose selection on an insect herbivore. We then measured herbivore survival as a function of three key phenotypic traits. We found that more traits were under selection in simpler vs. more complex food webs. The adaptive landscape was more neutral in complex food webs because different parasitoid species impose different selection pressures, minimizing relative fitness differences among phenotypes. Our results suggest that phenotypic evolution becomes more constrained in simplified food webs. This indicates that the simplification of ecological communities may constrain the adaptive potential of remaining populations to future environmental change. "What escapes the eye, however, is a much more insidious kind of extinction: the extinction of ecological interactions." Janzen (1974)

Phylogeny of Holarctic gall wasps of the genera Diplolepis and Periclistus (Hymenoptera: Cynipidae) based on DNA barcodes

Zhang, Y. M. — 2019-01-26

Rose gall wasps Diplolepis induce structurally distinct galls on wild roses, which provide gallers with food and shelter. These galls are attacked by a wide variety of micro-hymenopterans including another cynipid Periclistus that act as inquilines. Both Diplolepis and Periclistus are difficult to distinguish based on adult morphology, instead the structural appearance of galls is often used to distinguish species. Using the mitochondrial gene COI, we built phylogenies of both Diplolepis and Periclistus, while also estimating the ancestral host use of the inducers. Our phylogeny recovered the monophyly of Diplolepis, which have likely diverged from single-or multi-chambered leaf gallers to other plant organs. Periclistus exhibits a divide between the Palearctic and Nearctic clades, and ranges from specialists to generalists in terms of host specificity. The molecular results have largely supported the validity of species described in the literature, with notable exceptions in four species groups. While it is premature to enact any taxonomic changes without additional molecular markers, this incongruence between morphological and molecular data indicates these groups need taxonomic revision and gall morphology alone may be inadequate to delimit species.

Gene sharing networks to automate genome-based prokaryotic viral taxonomy

Jang, H. B. — 2019-01-29

Viruses of bacteria and archaea are likely to be critical to all natural, engineered and human ecosystems, and yet their study is hampered by the lack of a universal or scalable taxonomic framework. Here, we introduce vConTACT 2.0, a network-based application to establish prokaryotic virus taxonomy that scales to thousands of uncultivated virus genomes, and integrates confidence scores for all taxonomic predictions. Performance tests using vConTACT 2.0 demonstrate near-identical correspondence to the current official viral taxonomy (>85% genus-rank assignments at 96% accuracy) through an integrated distance-based hierarchical clustering approach. Beyond "known viruses", we used vConTACT 2.0 to automatically assign 1,364 previously unclassified reference viruses to tentative taxa, and scaled it to modern metagenomic datasets for which the reference network was robust to adding 16,000 viral contigs. Together these efforts provide a systematic reference network and an accurate, scalable taxonomic analysis tool that is critically needed for the research community.

Variation in space and time: a long-term examination of density-dependent dispersal in a woodland rodent

Denomme-Brown, S. T. — 2019-01-31

Dispersal is a fundamental ecological process that can be affected by population density, yet studies report contrasting effects of density on propensity to disperse. Additionally, the relationship between dispersal and density is seldom examined using densities measured at different spatial scales or over extensive time-series. We used 51-years of trapping data to examine how dispersal by wild deer mice (Peromyscus maniculatus) was affected by changes in both local and regional population densities. We examined these patterns over both the entire time-series and also in ten-year shifting windows to determine whether the nature and strength of the relationship changed through time. Probability of dispersal decreased with increased local and regional population density, and the negative effect of local density on dispersal was more pronounced in years with low regional densities. Additionally, the strength of negative density-dependent dispersal changed through time, ranging from very strong in some decades to absent in other periods of the study. Finally, while females were less likely to disperse, female dispersal was more density-dependent than male dispersal. Our study shows that the relationship between density and dispersal is not temporally static and that investigations of density-dependent dispersal should consider both local and regional population densities.

Non-Invasive Detection of Viral Antibodies Using Oral Flocked Swabs

Speicher, D. J. — 2019-01-31

Salivary antibodies are useful in surveillance and vaccination studies. However, low antibody levels and degradation by endonucleases are problematic. Oral flocked swabs are a potential non-invasive alternative to blood for detecting viral antibodies. Serum and saliva collected from 50 healthy volunteers were stored at -80{degrees}C; dried swabs at room temperature. Seroprevalence for Cytomegalovirus (CMV), Varicella-Zoster virus (VZV), Epstein-Barr virus (EBV), Measles and Mumps IgG antibodies were determined using commercial ELISAs and processed on an automated platform. For each antibody, swabs correlated well with saliva. For CMV IgG, VZV IgG, and EBV EBNA-1 IgG and VCA IgG, the swab sensitivities compared to serum were 95.8%, 96%, 92.1% and 95.5% respectively. For Measles IgG, swab sensitivity was 84.5%. Mumps IgG displayed poor sensitivity for oral swabs (60.5%) and saliva (68.2%). Specificities for IgG antibodies were 100% for CMV, EBV and Mumps. Specificities for VZV and Measles could not be determined due to seropositive volunteers. As oral flocked swabs correlate well with serum, are easy to self-collect and stable at room temperature further research is warranted. HighlightsO_LIOral flocked swabs are an easy, self-collection method for measuring viral antibodies. C_LIO_LIViral IgG is stable on dried oral flocked swabs for at least two years. C_LIO_LIOral swabs are highly sensitive for CMV, VZV, and EBV IgG. C_LIO_LIOral swabs are potentially useful for surveillance and clinical microbiology. C_LI

COI metabarcoding primer choice affects richness and recovery of indicator taxa in freshwater systems

Hajibabaei, M. — 2019-03-11

DNA-based biodiversity analysis has gained major attention due to the use of high throughput sequencing technology in approaches such as mixed community or environmental DNA metabarcoding. Many cytochrome c oxidase subunit I (COI) primer sets are now available for such work. The purpose of this study is to look at how COI primer choice affects the recovery of arthropod richness, beta diversity, and recovery of site indicator taxa in benthos kick-net samples typically used in freshwater biomonitoring. We examine 6 commonly used COI primer sets, on samples collected from 6 freshwater sites. Richness is sensitive to primer choice and the combined use of additional multiple COI amplicons recovers higher richness. Thus, to recover maximum richness, multiple primer sets should be used with COI metabarcoding. Samples consistently cluster by site regardless of amplicon choice or PCR replicate. Thus, for broadscale community analyses, overall beta diversity patterns are robust to COI marker choice. Additionally, the recovery of traditional freshwater bioindicator assemblages such as Ephemeroptera, Trichoptera, Plectoptera, and Diptera may not fully capture the diversity of broadscale arthropod site indicators that can be recovered from COI metabarcoding. Based on these results, studies that use different COI amplicons may not be directly comparable. This work will help future biodiversity and biomonitoring studies develop not just standardized, but optimized workflows that maximize taxon-detection or order taxa along gradients.

Watered-down biodiversity? A comparison of metabarcoding results from DNA extracted from matched water and bulk tissue biomonitoring samples

Hajibabaei, M. — 2019-03-12

Biomonitoring programs have evolved beyond the sole use of morphological identification to determine the composition of invertebrate species assemblages in an array of ecosystems. The application of DNA metabarcoding in freshwater systems for assessing benthic invertebrate communities is now being employed to generate biological information for environmental monitoring and assessment. A possible shift from the extraction of DNA from net-collected bulk benthic samples to its extraction directly from water samples for metabarcoding has generated considerable interest based on the assumption that taxon detectability is comparable when using either method. To test this, we studied paired water and benthos samples from a taxon-rich wetland complex, to investigate differences in the detection of taxa from each sample type. We demonstrate that metabarcoding of DNA extracted directly from water samples is a poor surrogate for DNA extracted from bulk benthic samples, focusing on key bioindicator groups. Our results continue to support the use of bulk benthic samples as a basis for metabarcoding-based biomonitoring, with nearly three times greater total richness in benthic samples compared to water samples. We also demonstrated that few arthropod taxa are shared between collection methods, with a notable lack of key bioindicator EPTO taxa in the water samples. Although species coverage in water could likely be improved through increased sample replication and/or increased sequencing depth, benthic samples remain the most representative, cost-effective method of generating aquatic compositional information via metabarcoding.

Studying ecosystems with DNA metabarcoding: lessons from aquatic biomonitoring

Bush, A. — 2019-03-18

An ongoing challenge for ecological studies has been the collection of data with high precision and accuracy at a sufficient scale to detect effects relevant to management of critical global change processes. A major hurdle for many workflows has been the time-consuming and challenging process of sorting and identification of organisms, but the rapid development of DNA metabarcoding as a biodiversity observation tool provides a potential solution. As high-throughput sequencing becomes more rapid and cost-effective, a big data revolution is anticipated, based on higher and more accurate taxonomic resolution, more efficient detection, and greater sample processing capacity. These advances have the potential to amplify the power of ecological studies to detect change and diagnose its cause, through a methodology termed Biomonitoring 2.0.nnDespite its promise, the unfamiliar terminology and pace of development in high-throughput sequencing technologies has contributed to a growing concern that an unproven technology is supplanting tried and tested approaches, lowering trust among potential users, and reducing uptake by ecologists and environmental management practitioners. While it is reasonable to exercise caution, we argue that any criticism of new methods must also acknowledge the shortcomings and lower capacity of current observation methods. Broader understanding of the statistical properties of metabarcoding data will help ecologists to design, test and review evidence for new hypotheses.nnWe highlight the uncertainties and challenges underlying DNA metabarcoding and traditional methods for compositional analysis, focusing on issues of taxonomic resolution, sample similarity, taxon misidentification, sample contamination, and taxon abundance. Using the example of freshwater benthic ecosystems, one of the most widely-applied non-microbial applications of DNA metabarcoding to date, we explore the ability of this new technology to improve the quality and utility of ecological data, recognising that the issues raised have widespread applicability across all ecosystem types.

Enhancing DNA metabarcoding performance and applicability with bait capture enrichment and DNA from conservative ethanol

Gauthier, M. — 2019-03-17

Metabarcoding is often presented as an alternative identification tool to compensate for coarse taxonomic resolution and misidentification encountered with traditional morphological approaches. However, metabarcoding comes with two major impediments which slow down its adoption. First, the picking and destruction of organisms for DNA extraction are time and cost consuming and do not allow organism conservation for further evaluations. Second, current metabarcoding protocols include a PCR enrichment step which induces errors in the estimation of species diversity and relative biomasses. In this study, we first evaluated the capacity of capture enrichment to replace PCR enrichment using controlled freshwater macrozoobenthos mock communities. Then, we tested if DNA extracted from the fixative ethanol (etDNA) of the same mock communities can be used as an alternative to DNA extracted from pools of whole organisms (bulk DNA). We show that capture enrichment provides more reliable and accurate representation of species occurrences and relative biomasses in comparison with PCR enrichment for bulk DNA. While etDNA does not permit to estimate relative biomasses, etDNA and bulk DNA provide equivalent species detection rates. Thanks to its robustness to mismatches, capture enrichment is already an efficient alternative to PCR enrichment for metabarcoding and, if coupled to etDNA, is a time-saver option in studies where presence information only is sufficient.

Fitness benefits of a motivated mom

Westrick, S. E. — 2019-03-31

Parental investment theory predicts that observed levels of parental care afforded to offspring are set by the benefits (to offspring quality and survival) relative to the costs (to parental survival or future reproduction). Although difficult to document in mammals, there is often substantial individual-variation in the amount of parental care within species. We measured the impact of individual variation in maternal care ("attentiveness" towards offspring or maternal motivation) on offspring growth and survival in a wild population of North American red squirrels (Tamiasciurus hudsonicus). We used latency to return to pups following a nest intrusion as a measure of maternal attentiveness to pups. We found this behavior to be repeatable within individuals suggesting this behavior is a personality trait or a "maternal style". In this population, postnatal growth rate is important for pup overwinter survival. Pups from large litters grew faster if they had a highly attentive mother, indicating that maternal care behavior can mitigate the trade-off between litter size and offspring growth and potentially improve survival of pups. Additionally, more attentive mothers had slightly higher lifetime reproductive success than less attentive mothers. These results highlight important fitness effects of having a highly attentive mother and show that maternal care behavior can alter a fundamental life history trade-off between offspring quantity and quality.nnLay SummaryIt pays to be attentive to your pups as a squirrel mom. In a long-term study of a wild population of North American red squirrels, we observed repeatable individual variation in maternal attentiveness towards offspring. Mothers who returned faster to pups following a nest intrusion produced faster growing pups and were able to produce larger fast-growing litters. Over their entire lifetime, attentive mothers also had more offspring recruit into the breeding population.

Translational control of breast cancer plasticity

Jewer, M. — 2019-04-02

Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5 Untranslated Regions (5UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. This leads to accumulation of corresponding proteins which induce plasticity and "fate-switching" toward stem-cell like phenotypes. Surprisingly, we observed that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem cell-like phenotypes. Strikingly, these effects can be overcome with drugs that antagonize translational reprogramming caused by eIF2 phosphorylation (e.g. ISRIB). Collectively, our findings unravel a hitherto unappreciated mechanism of induction of plasticity of breast cancer cells, and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.

Maternal stress promotes offspring growth without oxidative costs in wild red squirrels

Dantzer, B. — 2019-06-23

Elevations in glucocorticoid levels (GCs) in breeding females may induce adaptive shifts in offspring life histories. Offspring produced by mothers with elevated GCs may be better prepared to face harsh environments where a faster pace of life is beneficial. We examined how experimentally elevated GCs in pregnant or lactating North American red squirrels (Tamiasciurus hudsonicus) affected offspring postnatal growth, structural size, oxidative stress levels (two antioxidants and oxidative protein damage) in three different tissues (blood, heart, liver), and liver telomere lengths. We predicted that offspring from mothers treated with GCs would grow faster but would also have higher levels of oxidative stress and shorter telomeres, which may predict reduced longevity. Offspring from mothers treated with GCs during pregnancy were 8.3% lighter around birth but grew (in body mass) 17.0% faster than those from controls, whereas offspring from mothers treated with GCs during lactation grew 34.8% slower than those from controls and did not differ in body mass around birth. Treating mothers with GCs during pregnancy or lactation did not alter the oxidative stress levels or telomere lengths of their offspring. Fast-growing offspring from any of the treatment groups did not have higher oxidative stress levels or shorter telomere lengths, indicating that offspring that grew faster early in life did not exhibit oxidative costs after this period of growth. Our results indicate that elevations in maternal GCs may induce plasticity in offspring growth without long-term oxidative costs to the offspring that might result in a shortened lifespan. Summary StatementWe show that experimental increases in glucocorticoids in breeding female North American red squirrels affects offspring postnatal growth but not levels of oxidative damage and antioxidants or telomere lengths.

Ultra-Conserved Elements and morphology reciprocally illuminate conflicting phylogenetic hypotheses in Chalcididae (Hymenoptera, Chalcidoidea)

cruaud, a. — 2019-09-09

Recent technical advances combined with novel computational approaches promised the acceleration of our understanding of the tree of life. However, when it comes to hyperdiverse and poorly known groups of invertebrates, studies are still scarce. As published phylogenies will be rarely challenged by future taxonomists, careful attention must be paid to potential analytical bias. We present the first molecular phylogenetic hypothesis for the family Chalcididae, an emblematic group of parasitoid wasps, with a representative sampling (144 ingroups and 7 outgroups) that covers all described subfamilies and tribes and 82% of the known genera. Analyses of 538 Ultra-Conserved Elements (UCEs) with supermatrix (RAxML and IQTREE) and gene-tree reconciliation approaches (ASTRAL, ASTRID) resulted in highly supported topologies in overall agreement with morphology but reveal conflicting topologies for some of the deepest nodes. To resolve these conflicts, we explored the phylogenetic tree space with clustering and gene genealogy interrogation methods, analyzed marker and taxon properties that could bias inferences and performed a thorough morphological analysis (130 characters encoded for 40 taxa representative of the diversity). This joint analysis reveals that UCEs enable attainment of resolution between ancestry and convergent /divergent evolution when morphology is not informative enough, but also shows that a systematic exploration of bias with different analytical methods and a careful analysis of morphological features is required to prevent publication of artefactual results. We highlight a GC-content bias for ML approaches, an artefactual mid-point rooting of the ASTRAL tree and a deleterious effect of high percentage of missing data on gene tree reconciliation methods. Based on the results we propose a new classification of the family into eight subfamilies and 10 tribes that lay the foundation for future studies on the evolutionary history of Chalcididae.

Life out of water: Genomic and physiological mechanisms underlying skin phenotypic plasticity

Dong, Y.-w. — 2019-09-19

The Devonian radiation of vertebrates from aquatic into terrestrial habitats required behavioral, physiological, and morphological adaptations. Changes to skin structure and function were likely crucial, but adaptations were needed to resolve contrasting demands of maintaining a mechanical and physiological barrier while also facilitating ion and gas transport. Little is known of the mechanisms that underlie skin plasticity and adaptation between water and air. We performed experiments using two isogenic lineages of an amphibious killifish (Kryptolebias marmoratus from brackish and freshwater habitats) and used transcriptional and morphological data to reveal mechanisms recruited to resolve the dual challenges of skin providing both a barrier and an exchange interface during terrestrial acclimation. Transcriptional regulators of skin morphogenesis were quickly activated upon emersion. Regulation of cell-cell adhesion complexes, coupled with pathways homologous with those that regulate stratum corneum formation, was consistent with barrier function and mechanical reinforcement. Cutaneous respiration was associated with regulation of angiogenesis pathways and with blood vessel architecture that facilitated extremely short diffusion distances and direct delivery to ionocyotes. Evolutionary analyses revealed directional selection operating on proteins involved in barrier and respiratory functions, reinforcing the importance of these mechanisms for enabling the amphibious lifestyle of K. marmoratus. Fish from brackish niches were more resilient to emersion and also differed from freshwater fish in ionoregulatory responses to emersion. We conclude that plasticity of barrier, respiratory, and ionoregulatory functions in skin evolved to support the amphibious lifestyle of K. marmoratus; similar processes may have facilitated the terrestrial radiation of ancient fishes.nnSignificance statementThe transition of vertebrate life from water to land coincided with solving multiple physiological challenges including avoiding drying out while also exchanging gases and ions with the environment. Though changes in the skin were likely important, little is known of the mechanisms that underlie skin flexibility and adaptation between water and air. We performed air exposure experiments with an amphibious killifish; gene expression profiling, microscopy, and evolutionary analysis of proteins revealed cell structures, proteins, and molecular pathways that support skin flexibility and adaptations during air exposure, and ion regulation contributed to differences in killifish abilities to adjust to air. Amphibious killifish are useful models to help us understand changes that enable water to air transitions in contemporary and ancient fishes.

Activating M1 muscarinic cholinergic receptors induces destabilization of resistant contextual fear memories in rats

Abouelnaga, K. H. — 2023-05-18

Destabilization of previously consolidated memories places them in a labile state in which they are open to modification. However, strongly encoded fear memories tend to be destabilization-resistant and the conditions required to destabilize such memories remain poorly understood. Our lab has previously shown that exposure to salient novel contextual cues during memory reactivation can destabilize strongly encoded object location memories and that activity at muscarinic cholinergic receptors is critical for this effect. In the current study, we similarly targeted destabilization-resistant fear memories, hypothesizing that exposure to salient novelty at the time of reactivation would induce destabilization of strongly encoded fear memories in a muscarinic receptor-dependent manner. First, we show that contextual fear memories induced by 3 context-shock pairings readily destabilize upon memory reactivation, and that this destabilization is blocked by systemic (ip) administration of the muscarinic receptor antagonist scopolamine (0.3mg/kg) in male rats. Next, we demonstrate that more strongly encoded fear memories (induced with 5 context-shock pairings) resist destabilization. Consistent with our previous work, however, we report that salient novelty (a change in floor texture) presented during the reactivation session promotes destabilization of resistant contextual fear memories in a muscarinic receptor-dependent manner. Finally, the effect of salient novelty on memory destabilization was mimicked by stimulating muscarinic receptors with the selective M1 agonist CDD-0102A (ip, 0.3mg/kg). These findings reveal further generalizability of our previous results implicating novel cues and M1 muscarinic signaling in promoting destabilization of resistant memories and suggest possible therapeutic options for disorders characterized by persistent, maladaptive fear memories such as PTSD and phobias.

A chemical genetic screen uncovers novel seed priming agents capable of persistent perturbation of anthocyanin regulation in Arabidopsis thaliana

Hiiback, K. M. — 2023-05-23

Priming is a general term for a phenomenon in which exposure to an early environmental stimulus results in a more rapid or vigorous response when the plant is exposed to subsequent challenges. Various types of priming have been described: systemic acquired resistance against biotic stimuli, epigenetic stress memory induced by repeated abiotic stimuli, and seed priming treatments which apply water and additional adjuncts to seeds to improve crop performance. Using a high-throughput chemical genomic approach, thousands of small molecules were screened to identify compounds capable of chemical priming, asking if these could serve as artificial environments to persistently induce altered response to later abiotic challenges when applied as seed treatments. Attenuation of expected visual anthocyanin accumulation was chosen as a screening phenotype due to the visual nature of these pigments, as well as their biological roles in development and stress response. Several novel structural categories of molecules were identified that had the ability to reduce total anthocyanin accumulation in 7-18-day old seedlings induced by later low temperature challenge, persisting days after the removal of the compounds. Application variables were explored with thought to future use of the priming compounds as functional treatments: a dose-dependent relationship was established, additional effects on growth and development were documented to ensure minimal detrimental side effects on the treated plants, and the necessary temporal window of treatment was explored and reduced. Cross testing of the priming treatments identified by low temperature screening for ability to reduce anthocyanin induction by alternative exogenous and endogenous conditions showed consistent attenuative effect and revealed that the effect of priming on this metabolic phenotype was not specific to low temperature response. The presented research represents a proof-of-concept for the functional potential of seed priming with novel compounds, and highlights anthocyanin accumulation as a flexible component of plant stress response.

Soybean-SCN duel: Novel insight into Soybean's Resistant Responses to Heterodera glycines

Torabi, S. — 2023-05-23

Soybean cyst nematodes (SCN, Heterodera glycines Ichinohe) are a significant threat to soybean production globally, causing severe yield losses and necessitating the development of effective strategies to combat this devastating nematode disease. This study presents dual RNA-seq analyses of the three most common SCN-resistant lines (Plant Introduction (PI) 437654, 548402, and 88788) and the susceptible line Lee74 against the SCN HG type 1.2.5.7 to identify the mechanisms of resistance and virulence genes involved in resistance breakdown. Transcriptomic and pathway analyses reveal the activation of the phenylpropanoid pathway, MAPK signaling pathway, plant hormone signal transduction, and secondary metabolite pathways in the resistance mechanisms. PI 437654, which exhibited robust resistance (female index, FI=0%), demonstrated unique gene expression associated with cell wall reinforcement, oxidative enzymes, ROS scavengers, and Ca+2 sensors governing the salicylic acid (SA) biosynthesis process, indicating its key defense mechanism. Moreover, using different hosts with varying levels of immunity and a susceptible line provided insights into SCN pathogenesis and how H. glycine overcomes different layers of host immunity by modulating its virulence genes. This research provides novel insights into the molecular mechanisms underlying soybean-SCN interactions and identifies potential targets for developing strategies to manage this devastating nematode disease.

Ultrasonographic measurements of fascicle length overestimate adaptations in serial sarcomere number

Hinks, A. — 2023-06-06

Ultrasound-derived measurements of muscle fascicle length (FL) are often used to infer increases (chronic stretch or training) or decreases (muscle disuse or aging) in serial sarcomere number (SSN). Whether FL adaptations measured via ultrasound can truly approximate SSN adaptations has not been investigated. We casted the right hindlimb of 15 male Sprague-Dawley rats in a dorsiflexed position (i.e., stretched the plantar flexors) for 2 weeks, with the left hindlimb serving as a control. Ultrasound images of the soleus, lateral gastrocnemius (LG), and medial gastrocnemius (MG) were obtained with the ankle at 90{degrees} and full dorsiflexion for both hindlimbs pre and post-cast. Following post-cast ultrasound measurements, legs were fixed in formalin with the ankle at 90{degrees}, then muscles were dissected, and fascicles were teased out for measurement of sarcomere lengths via laser diffraction and calculation of SSN. Ultrasound detected an 11% increase in soleus FL, a 12% decrease in LG FL, and an 8-11% increase in MG FL for proximal fascicles and at full dorsiflexion. These adaptations were partly reflected by SSN adaptations, with a 6% greater soleus SSN in the casted leg than the un-casted leg, but no SSN differences for the gastrocnemii. Weak relationships were observed between ultrasonographic measurements of FL and measurements of FL and SSN from dissected fascicles. Our results showed that ultrasound-derived FL measurements can overestimate an increase in SSN by [~]5%. Future studies should be cautious when concluding a large magnitude of sarcomerogenesis from ultrasound-derived FL measurements, and may consider applying a correction factor. Key Points SummaryO_LIMeasurements of muscle fascicle length via ultrasound are often used to infer changes in serial sarcomere number, such as increases following chronic stretch or resistance training, and decreases with aging or muscle disuse C_LIO_LIThe present study used a rat model of casting the plantar flexor muscles in a stretched position to investigate directly whether ultrasound-derived fascicle length can accurately detect adaptations in serial sarcomere number C_LIO_LIUltrasound detected an [~]11% increase in soleus fascicle length, but measurements on dissected fascicles showed the actual increase in serial sarcomere number was only [~]6%; therefore, measurements of ultrasound-derived fascicle length can overestimate serial sarcomere number adaptations by as much as 5% C_LI

Intraspecific genetic variation is critical to robust toxicological predictions in Daphnia

Shahmohamadloo, R. S. — 2023-06-07

Environmental risk assessment is a critical tool for protecting aquatic life and its effectiveness is predicated on predicting how natural populations respond to contaminants. Yet, routine toxicity testing typically examines only one genotype, which may render risk assessments inaccurate as populations are most often composed of genetically distinct individuals. To determine the importance of intraspecific variation in the translation of toxicity testing to populations, we quantified the magnitude of genetic variation within 20 Daphnia magna clones derived from one lake using whole genome sequencing and phenotypic assays. We repeated these assays across two exposure levels of microcystins, a cosmopolitan and lethal aquatic contaminant produced by harmful algal blooms. We found considerable intraspecific genetic variation in survival, growth, and reproduction, which was amplified by microcystins exposure. Finally, using simulations we demonstrate that the common practice of employing a single genotype to calculate toxicity tolerance failed to produce an estimate within the 95% confidence interval over half of the time. These results illuminate the importance of incorporating intraspecific genetic variation into toxicity testing to reliably predict how natural populations will respond to aquatic contaminants.

Cell morphology best predicts tumorigenicity and metastasis in vivo across multiple TNBC cell lines of different metastatic potential

Conner, S. J. — 2023-06-14

BackgroundMetastasis is the leading cause of death in breast cancer patients. For metastasis to occur, tumor cells must invade locally, intravasate, and colonize distant tissues and organs, all steps that require tumor cell migration. The majority of studies on invasion and metastasis rely on human breast cancer cell lines. While it is known that these cells have different properties and abilities for growth and metastasis, the in vitro morphological, proliferative, migratory, and invasive behavior of these cell lines and their correlation to in vivo behavior is poorly understood. Thus, we sought to classify each cell line as poorly or highly metastatic by characterizing tumor growth and metastasis in a murine model of six commonly used human triple-negative breast cancer xenografts, as well as determine which in vitro assays commonly used to study cell motility best predict in vivo metastasis. MethodsWe evaluated the liver and lung metastasis of human TNBC cell lines MDA-MB-231, MDA-MB-468, BT549, Hs578T, BT20, and SUM159 in immunocompromised mice. We characterized each cell lines cell morphology, proliferation, and motility in 2D and 3D to determine the variation in these parameters between cell lines. ResultsWe identified MDA-MB-231, MDA-MB-468, and BT549 cells as highly tumorigenic and metastatic, Hs578T as poorly tumorigenic and metastatic, BT20 as intermediate tumorigenic with poor metastasis to the lungs but highly metastatic to the livers, and SUM159 as intermediate tumorigenic but poorly metastatic to the lungs and livers. We showed that metrics that characterize cell morphology are the most predictive of tumor growth and metastatic potential to the lungs and liver. Further, we found that no single in vitro motility assay in 2D or 3D significantly correlated with metastasis in vivo. ConclusionsOur results provide an important resource for the TNBC research community, identifying the metastatic potential of 6 commonly used cell lines. Our findings also support the use of cell morphological analysis to investigate the metastatic potential and emphasize the need for multiple in vitro motility metrics using multiple cell lines to represent the heterogeneity of metastasis in vivo.

Can higher-order interactions resolve the species coexistence paradox?

Gibbs, T. L. — 2023-06-23

Most ecological models are based on the assumption that species interact in pairs. Emerging in diverse communities, however, are higher-order interactions, in which two or more species jointly impact the growth of a third species. A pitfall of the pairwise approach is that it misses the higher-order interactions potentially responsible for maintaining diversity in nature. Here, we explore how well higher-order interactions enable coexistence when pairwise interactions are insufficient to do so. Specifically, we explore the stability properties of systems where higher-order interactions guarantee that a specified set of abundances are a feasible equilibrium of the dynamics. Even these higher-order interactions do not necessarily produce robust coexistence. We find that facilitative higher-order interactions that counter pairwise competitive interactions are less likely to generate stable equilibria than competitive interactions countering pairwise facilitation. In both cases the system is more likely to be stable when the pairwise interactions are weak, and when the equilibrium abundances are near those produced by the pairwise interactions. Last, we show that correlations between the pairwise and higher-order interactions permit robust coexistence in diverse systems. Our work reveals the challenges in generating stable coexistence through higher-order interactions, but also uncovers higher-order interaction patterns that can enable diversity.

Paranannizziopsis spp. associated with skin lesions in wild snakes in North America and development of a real-time PCR assay for rapid detection of the fungus in clinical samples

Lorch, J. M. — 2023-07-13

The emergence of ophidiomycosis (or snake fungal disease) in snakes has prompted increased awareness of the potential effects of fungal infections on wild reptile populations. Yet, aside from Ophidiomyces ophidiicola, little is known about other mycoses affecting wild reptiles. The closely related genus Paranannizziopsis has been associated with dermatomycosis in snakes and tuataras in captive collections, and P. australasiensis was recently identified as the cause of skin infections in non-native wild panther chameleons (Furcifer pardalis) in Florida, USA. Here we describe five cases of Paranannizziopsis spp. associated with skin lesions in wild snakes in North America and one additional case from a captive snake from Connecticut, USA. In addition to demonstrating that wild Nearctic snakes can serve as a host for these fungi, we also provide evidence that the genus Paranannizziopsis is widespread in wild snakes, with cases being identified in Louisiana (USA), Minnesota (USA), Virginia (USA), and British Columbia (Canada). Phylogenetic analyses conducted on multiple loci of the fungal strains we isolated identified P. australasiensis in Louisiana and Virginia; the remaining strains from Minnesota and British Columbia did not cluster with any of the described species of Paranannizziopsis, although the strains from British Columbia appear to represent a single lineage. Finally, we designed a pan-Paranannizziopsis real-time PCR assay targeting the internal transcribed spacer region 2. This assay successfully detected DNA of all described species of Paranannizziopsis and the two potentially novel taxa isolated in this study and did not cross-react with closely related fungi or other fungi commonly found on the skin of snakes. The assay was 100% sensitive and specific when screening clinical (skin tissue or skin swab) samples, although full determination of the assays performance will require additional follow up due to the small number of clinical samples (n=14 from 11 snakes) available for testing in our study. Nonetheless, the PCR assay can provide an important tool in further investigating the prevalence, distribution, and host range of Paranannizziopsis spp. and facilitate more rapid diagnosis of Paranannizziopsis spp. infections that are otherwise difficult to differentiate from other dermatomycoses.

A high-quality reference genome for the common creek chub, Semotilus atromaculatus

Meuser, A. V. — 2023-07-15

Creek chub (Semotilus atromaculatus) are a leuciscid minnow species commonly found in anthropogenically disturbed environments, making them an excellent model organism to study human impacts on aquatic systems. Genomic resources for creek chub and other leuciscid species are currently limited. However, advancements in DNA sequencing now allow us to create genomic resources at a historically low cost. Here, we present a high quality 239 contig reference genome for the common creek chub, created with PacBio HiFi sequencing. We compared the assembly quality of two pipelines: Pacific Biosciences Improved Phase Assembly (IPA; 873 contigs) and Hifiasm (239 contigs). Quality and completeness of this genome is comparable to the zebrafish (Danioninae) and fathead minnow (Leuciscidae) genomes. The creek chub genome is highly syntenic to the zebrafish and fathead minnow genomes, and while our assembly does not resolve into the expected 25 chromosomes, synteny with zebrafish suggests that each creek chub chromosome is likely represented by 1-4 large contigs in our assembly. This reference genome is a valuable resource that will enhance genomic bio-diversity studies of creek chub and other non-model leuciscid species common to disturbed environments.

The Effect of Gradual Ovarian Failure on Dynamic Muscle Function and the Role of High Intensity Interval Training on Mitigating Impairments

Hubbard, E. F. — 2023-07-17

Skeletal muscle function is impaired in menopause and exercise may mitigate tshis decline. We used the VCD model of menopause to investigate the effects of gradual ovarian failure on skeletal muscle contractile function and whether high intensity interval training (HIIT) can mitigate impairments. Sexually mature female CD-1 mice were assigned to one of three groups: control (n=5), VCD-sedentary (n=5), or VCD-training (n=5). Following ovarian failure, the VCD-training group underwent 8 weeks of uphill HIIT. Mice were sacrificed 8 weeks after ovarian failure, representing late menopause. Single muscle fibres from the soleus (SOL) and extensor digitorum longus (EDL) muscles were dissected, chemically permeabilized, and mechanically tested. Single muscle fibres were maximally activated (pCa 4.5) then isotonic load clamps were performed to calculate force-velocity-power curves. Absolute force and peak power were 31% and 32% lower in VCD-sedentary fibres compared to control fibres, respectively, in both SOL and EDL muscles. Despite reductions in absolute force and therefore lighter relative loads imposed during the isotonic contractions in VCD-sedentary fibres, there were no concomitant increases in contractile velocity. HIIT was partially effective at mitigating power loss (22% higher peak power in VCD-training compared to VCD-sedentary), but only in fast-type SOL fibres. These findings indicate that ovarian failure impairs dynamic contractile function - likely through a combination of lower force-generating capacity and slower shortening velocity - and that HIIT may be insufficient to completely counteract the deleterious effects of menopause at the cellular level. New & NoteworthyO_LIReductions in circulating ovarian hormones impair static muscle contractile performance, but less is known about dynamic properties like power. C_LIO_LITypically, rodent models of menopause completely remove the ovaries and fail to mimic the prolonged and complex hormonal transition that includes a retention in ovarian androgen production. C_LIO_LIUsing an ovary intact VCD model of ovarian failure, we found that single fibre power was impaired compared with controls in both SOL and EDL fibres. C_LIO_LIOur uphill high intensity interval training program was partially sufficient to reverse power loss, but only in fast-type SOL fibres. C_LIO_LIImpairments in muscle power following ovarian failure are likely driven by a combination of decreased muscle size and force-generating capacity. C_LI

OSGN-1 is a conserved flavin-containing monooxygenase required to stabilize the intercellular bridge in late cytokinesis

Goupil, E. — 2023-08-07

Cytokinesis is the last step of cell division and is regulated by the small GTPase RhoA. RhoA activity is required for all steps of cytokinesis, including prior to abscission when daughter cells are ultimately physically separated. Like germ cells in all animals, the C. elegans embryonic germline founder cell initiates cytokinesis but does not complete abscission, leaving a stable intercellular bridge between the two daughter cells. Here we identify and characterize C. elegans OSGN-1 as a novel cytokinetic regulator that promotes RhoA activity during late cytokinesis. Sequence analyses and biochemical reconstitutions reveal that OSGN-1 is a flavin-containing monooxygenase. Genetic analyses indicate that the monooxygenase activity of OSGN-1 is required to maintain active RhoA at the end of cytokinesis in the germline founder cell and to stabilize the intercellular bridge. Deletion of OSGIN1 in human cells results in an increase in binucleation as a result of cytokinetic furrow regression, and this phenotype can be rescued by expressing a catalytically-active form of C. elegans OSGN-1, indicating that OSGN-1 and OSGIN1 are functional orthologs. We propose that OSGN-1 and OSGIN1 are novel, conserved monooxygenase enzymes required to maintain RhoA activity at the intercellular bridge during late cytokinesis and thus promote its stability, enabling proper abscission in human cells and bridge stabilization in C. elegans germ cells.

BirT: a novel primer pair for avian environmental DNA metabarcoding

Thalinger, B. — 2023-08-09

Environmental DNA metabarcoding has become a widely used technique to detect animals from environmental samples and is on the brink of being implemented into routine species monitoring. Surprisingly, birds are among the taxonomic groups which until recently, received comparably little attention in eDNA research, a fact that is changing rapidly, with the growing number of air eDNA analyses. Since birds are hardly ever the most abundant species in aquatic or terrestrial habitats, a high specificity of the employed metabarcoding primers is key to limit non-target amplifications and enable reliable detection from environmental samples. Here, we present a novel primer pair (BirT) for metabarcoding of avian environmental DNA. We optimized specificity and fragment length regarding taxonomic resolution and available sequencing technology. Additionally, we evaluated the availability of 12S reference sequences for birds and filled database gaps by generating novel 12S barcodes. Finally, we tested the applicability of this approach using field-collected eDNA samples obtained with three different filter types. These results were compared to visual observations uploaded to eBird (www.eBird.org) during the sampling period. Our results confirm the suitability of the BirT primer pair for bird eDNA metabarcoding with optimized fragment length, no amplification of key non-target groups, and taxonomic resolution provided by the amplified fragment. Albeit there are still substantial gaps in the 12S reference sequence database, the analysis of bird eDNA from water samples resulted in species-level taxonomic resolution for 92% of the detected taxa. All tested filter/filtration combinations delivered similar results for total read numbers per sample (mean: 613,972 {+/-} 340,088 SD) and species detected per sample (mean: 5.5 {+/-} 2.3 SD). Ninety-five percent of the bird detections were highly plausible and 58% confirmed by visual observations. The majority of the detected bird species was closely associated with aquatic habitats confirming the suitability of water samples for the detection of waterfowl and species inhabiting similar ecological niches via eDNA.

Comparative Restriction Enzyme Analysis of Methylation (CREAM) Reveals Methylome Variability Within a Clonal In Vitro Cannabis Population

Boissinot, J. — 2023-08-19

The primary focus of medicinal cannabis research is to ensure the stability of cannabis lines for consistent administration of chemically consistent products to patients. In recent years, tissue culture has emerged as a valuable technique for genetic preservation and rapid production of cannabis clones. However, there is concern that the physical and chemical conditions of the growing media can induce somaclonal variation, potentially impacting the viability and uniformity of clones. To address this concern, we developed Comparative Restriction Enzyme Analysis of Methylation (CREAM), a novel method to assess DNA methylation patterns and used it to assess a population of 78 cannabis clones maintained in tissue culture. Through bioinformatics analysis of the methylome, we successfully detected 2,272 polymorphic methylated regions among the clones. Remarkably, our results demonstrated that DNA methylation patterns were preserved across subcultures within the clonal population, allowing us to distinguish between two subsets of clonal lines used in this study. These findings significantly contribute to our understanding of the epigenetic variability within clonal lines in medicinal cannabis produced through tissue culture techniques. This knowledge is crucial for understanding the effects of tissue culture on DNA methylation and ensuring the consistency and reliability of medicinal cannabis products with therapeutic properties. Additionally, the CREAM method is a fast and affordable technology to get a first glimpse at methylation in a biological system. It offers a valuable tool for studying epigenetic variation in other plant species, thereby facilitating broader applications in plant biotechnology and crop improvement.

Zebrafish acta1b as a Candidate for Modelling Human Actin Cardiomyopathies

Prill, K. — 2023-08-30

Heart failure is the leading cause of mortality worldwide, primarily associated with cardiovascular disease. Many heart muscle diseases are caused by mutations in genes that encode contractile proteins, including cardiac actin mutations. Zebrafish are an advantageous system for modelling cardiac diseases due to their ability to develop without a functional heart throughout embryonic development. However, genome duplication in the teleost lineage poses a unique challenge by increasing the number of genes involved in heart development. Four actin genes are expressed in the zebrafish heart: acta1b, actc2, and duplicates of actc1a on chromosomes 19 and 20. In this study, we characterize the actin genes involved in early zebrafish heart development using in situ hybridization and CRISPR targeting to determine the most suitable gene for modelling actin changes observed in human patients with heart disease. The actc1a and acta1b genes are predominantly expressed during embryonic heart development, resulting in severe cardiac phenotypes when targeted with CRISPR. Considering the duplication of the actc1a gene, we recommend acta1b as the best gene for targeted cardiac actin research.

Evolutionary diversity of the control of the azole response by Tra1 across yeast species

Librais, G. M. — 2023-09-06

Tra1 is an essential co-activator protein of the yeast SAGA and NuA4 acetyltransferase complexes that regulate gene expression through multiple mechanisms including the acetylation of histone proteins. Tra1 is a pseudokinase of the PIKK family characterized by a C-terminal PI3K domain with no known kinase activity. However, mutations of specific arginine residues to glutamine in the PI3K domains (an allele termed tra1Q3) result in reduced growth and increased sensitivity to multiple stresses. In the opportunistic fungal pathogen Candida albicans, the tra1Q3 allele reduces pathogenicity and increases sensitivity to the echinocandin antifungal drug caspofungin, which disrupts the fungal cell wall. Here, we found that loss of Tra1 function, in contrast to what is seen with caspofungin, increases tolerance to the azole class of antifungal drugs, which inhibits ergosterol synthesis. In C. albicans, tra1Q3 increases expression of genes linked to azole resistance, such as ERG11 and CDR1. CDR1 encodes a multidrug ABC transporter associated with efflux of multiple xenobiotics, including azoles. Consequently, cells carrying tra1Q3 show reduced intracellular accumulation of fluconazole. In contrast, a tra1Q3 S. cerevisiae strain displayed opposite phenotypes: decreased tolerance to azole, decreased expression of the efflux pump PDR5 and increased intracellular accumulation of fluconazole. Therefore, our data provide evidence that Tra1 differentially regulates the antifungal response across yeast species.

Autophagy-dependent alternative splicing event produces a more stable ribosomal protein S24 isoform that aids in hypoxic cell survival

Kerry, J. — 2023-09-25

Overlapping or convergent stress-activated molecular pathways exist to coordinate cell fate in response to stimuli such as hypoxia, oxidative stress, DNA damage, and unfolded proteins. Cells can remodel the splicing and translation machineries to mount a specialized gene expression response to certain stresses. Here, we show that hypoxic human cells in 2D and 3D culture models increase the relative abundance by 1.7- to 2.6-fold and 4.7- to 11.5-fold, respectively, of a longer mRNA variant of ribosomal protein S24 (RPS24L) compared to a shorter mRNA variant (RPS24S) by favoring the inclusion of a 22 bp cassette exon. Mechanistically, RPS24L and RPS24S are induced and repressed, respectively, by distinct parallel pathways in hypoxia: RPS24L is induced in an autophagy-dependent manner, while RPS24S is reduced by mTORC1 repression and in a HIF-dependent manner. RPS24L is a more stable mRNA in hypoxia and produces a more stable protein isoform compared to RPS24S. Cells overexpressing RPS24L display improved survival and growth in hypoxia relative to control cells and cells overexpressing RPS24S, which display impaired survival. Previous work from our group showed a correlation between RPS24L levels and tumor hypoxia in prostate cancer. These data highlight RPS24L as a stress-induced alternative splicing event that favors hypoxic cell survival, which could be exploited by cancer cells in the tumor microenvironment.

Cardiac atrophy, dysfunction, and metabolic impairments: a cancer-induced heart failure phenotype

Ogilvie, L. M. — 2023-10-02

Muscle atrophy and weakness are prevalent features of cancer. While extensive research has characterized skeletal muscle wasting in cancer cachexia, limited studies have investigated how cardiac structure and function are affected by therapy-naive cancer. In cell-based models of orthotopic, syngeneic epithelial ovarian cancer (EOC) and pancreatic ductal adenocarcinoma (PDAC), and a patient-derived pancreatic xenograft model (PDX), we evaluated cardiac structure, function, and metabolism. Tumor-bearing mice showed cardiac atrophy and intrinsic systolic and diastolic dysfunction; associated with hypotension and exercise intolerance. In hearts of ovarian tumor-bearing mice, fatty acid-supported mitochondrial respiration decreased and carbohydrate-supported respiration increased, establishing a substrate shift in cardiac metabolism that is characteristic of heart failure. EOC decreased cytoskeletal and cardioprotective gene expression, which was paralleled by downregulation of transcription factors that regulate cardiomyocyte size and function. PDX tumors altered myosin heavy chain isoform expression - a molecular phenotype observed in heart failure. Markers of autophagy and ubiquitin-proteasome system were upregulated with cancer, providing evidence of catabolic signaling that promotes cardiac wasting. Together, metabolic stress, cardiac gene dysregulation, and upregulation of catabolic pathways contribute to cardiac atrophy and failure during cancer. Finally, we demonstrate that pathological cardiac remodeling is induced by human cancer, providing translational evidence of cancer-induced cardiomyopathy.

A highly resolved multiplex network reveals the structural role of insects and plants in terrestrial food webs

Hale, K. R. S. — 2023-10-07

Comparative studies suggest remarkable similarities among food webs across habitats, including systematic changes in their structure with diversity and complexity (scale-dependence). However, historic aboveground terrestrial food webs (ATFWs) have coarsely grouped plants and insects such that these webs are generally small, and herbivory is disproportionately underrepresented compared to vertebrate predator-prey interactions. Furthermore, terrestrial herbivory is thought to be structured by unique processes compared to size-structured feeding in other systems. Here, we present the richest ATFW to date, including [~]580,000 feeding links among [~]3,800 taxonomic species, sourced from [~]27,000 expert-vetted interaction records annotated as feeding upon one of six different resource types: leaves, flowers, seeds, wood, prey, and carrion. By comparison to historical ATFWs and null ecological hypotheses, we show that our temperate forest web displays a potentially unique structure characterized by two properties: a) a large fraction of carnivory interactions dominated by a small number of hyper-generalist, opportunistic bird and bat predators, and b) a smaller fraction of herbivory interactions dominated by a hyper-rich community of insects with variably-sized but highly-specific diets. We attribute our findings to the large-scale, even resolution of vertebrate, insect, and plant guilds in our food web.

FARESHARE: An Open-source Apparatus for Assessing Drinking Microstructure in Socially Housed Rats

Frie, J. A. — 2023-10-09

Social factors have been shown to play a significant and lasting role in alcohol consumption. Studying the role of social context on alcohol drinking is important to understand the factors that contribute to initiation or maintenance of casual and problematic alcohol use, as well as those that may be protective. A substantial body of preclinical research has shown that social environment such as housing conditions and social rank plays an important role in alcohol consumption and preference, though the extent of these effects have been obfuscated by methodological differences and technical challenges. Robust individual differences in alcohol intake in socially housed animals are difficult to track when animals share a common fluid source. Commercial solutions are prohibitively expensive and are limited by proprietary software and hardware (including caging systems). Here we describe an affordable, open-source solution for tracking fluid consumption in socially housed rats.

Reduced S-acylation of SQSTM1/p62 in Huntington disease is associated with impaired autophagy

Abrar, F. — 2023-10-11

Disruption of macroautophagy/autophagy has emerged as a common feature in many neurodegenerative diseases. Autophagy is a membrane-dependent pathway that requires many key regulators to quickly localize on and off membranes during induction promoting membrane fusion. Previously, our bioinformatic approaches have shown that autophagy and Huntington disease (HD) are enriched in S-acylated proteins. S-acylation involves the reversible addition of long chain fatty acids to promote membrane binding. Herein, we show that inhibition of S-acylation regulates the abundance of several key regulators of autophagy and leads to a partial block of autophagic flux. We show that the autophagy receptor SQSTM1/p62 (sequestosome 1) is S-acylated and directed to the lysosome. Importantly, we see that SQSTM1 S-acylation is significantly reduced in HD patient and mouse model brains, thus providing a novel mechanism for the generation of empty autophagosomes previously seen in HD models and patient cells.

Contrasting patterns of presence-absence variation of NLRS within S. chilense are mainly shaped by past demographic history

Silva Arias, G. A. — 2023-10-17

Understanding the evolution of pathogen resistance genes (nucleotide-binding site-leucine-rich repeats, also known as NLRs) within a species requires a comprehensive examination of factors that affect gene loss and gain. We present a new reference genome of Solanum chilense, that leads to an increased number and more accurate annotation of NLRs. Next, using a target-capture approach, we quantify the presence-absence variation (PAV) of NLR loci across 20 populations from different habitats. We build a rigorous pipeline to validate the identification of PAV of NLRs, then show that PAV is larger within populations than between populations, suggesting that maintenance of NLR diversity is linked to population dynamics. Furthermore, the amount of PAV is not correlated with the NLR presence in gene clusters in the genome, but rather with the past demographic history of the species, with loss of NLRs in diverging populations at the distribution edges and smaller population sizes. Finally, using a redundancy analysis, we find limited evidence of PAV being linked to environmental gradients. Our results contradict the classic assumptions of the important selective role of PAV for NLRs, and suggest that NLRs PAV is driven by random processes (and weak selection) in an outcrossing plant with high nucleotide diversity.

Effects of intensified training with insufficient recovery on joint level and single muscle fibre mechanical function: The role of myofibrillar Ca2+ sensitivity

Roussel, O. — 2023-10-17

Intense exercise training with insufficient recovery is associated with reductions in neuromuscular performance. However, it is unclear how single muscle fibre mechanical function and myofibrillar Ca2+ sensitivity contribute to these impairments. We investigated the effects of overload training on joint-level neuromuscular performance and cellular-level mechanical function. Fourteen athletes (4 female, 10 male) underwent a 3-week intensified training protocol consisting of [~]140% of their regular training hours with three additional high-intensity training sessions per week. Neuromuscular performance of the knee extensors was assessed via maximum voluntary contraction (MVC) force, electrically evoked twitch contractions, and a force-frequency relationship. Muscle biopsies were taken from the vastus lateralis to assess single fibre mechanical function. Neither MVC force nor twitch parameters were altered following intensified training (all p>0.05), but a rightward shift in the force-frequency curve was observed with a 6-27% reduction in force at low-frequencies (5-20Hz, all p<0.05). In single fibres, maximal force output was not reduced following intensified training, but there was a rightward shift in the force-pCa curve driven by a 6% reduction in Ca2+ sensitivity as indicated by a lower pCa50 value (i.e., higher [Ca2+]) across fibre types (Pre=6.477{+/-}0.157, Post=6.088{+/-}0.480, p<0.05). These data indicate intensified training leads to impaired Ca2+ sensitivity at the single fibre level, which in part explains impaired neuromuscular function at the joint level during lower frequencies of activation. This is an important consideration for athletes, as performance is often assessed at maximal levels of activation, and these underlying impairments in force generation may be less obvious. New & NoteworthyIntense exercise training with insufficient recovery leads to impaired muscle contractile performance. These impairments often manifest at lower frequencies of muscle stimulation, termed prolonged low-frequency force depression. Impaired myofibrillar calcium sensitivity has been suggested as a potential mechanism of prolonged low-frequency force depression. Our work shows that impaired calcium sensitivity of single muscle fibres coincided with joint level prolonged low-frequency force depression after intense exercise training with insufficient recovery.

Protocols to enable fluorescence microscopy of microbial interactions on living maize silks (style tissue) that balance the requirements for containment, biological relevance, viability, and experimental accessibility

Thompson, M. E. H. — 2023-11-01

There is interest in studying microbes that colonize maize silks (style tissue, critical for reproduction) including the fungal pathogen Fusarium graminearum (Fg) and its interactions with the microbiome and biocontrol agents. In planta imaging of these interactions on living silks using confocal fluorescence microscopy would provide key insights. However, newly discovered microbes have unknown effects of on human health, and there are regulatory requirements to prevent the release of fluorescently-tagged microbes into the environment. Therefore, the microbe infection, colonization, and interaction stages on silks prior to microscopy must be contained. At the same time, silk viability must be maintained and experiments conducted that are biologically relevant (e.g. silks should remain attached to the cob), yet the silk tissue must be accessible to the researcher (i.e. not within husk leaves) and allow for multiple replicates. Here we present methods that meet these five contrasting criteria. We tested these methods using Fg and four silk-derived bacterial endophytes. The endophytes were previously known to have anti-Fg activity in vitro, but in planta observations were lacking. In Method 1, a portion of the tip of a cob was dissected, and silks remained attached to the cob in a Petri dish. The cob was placed on a water agar disc to maintain hydration. DsRed-tagged bacteria and GFP-tagged Fg were inoculated onto the silks and incubated, allowing the two microbes to grow towards one another before staining with propidium iodide for confocal microscopy. A variation of the protocol was presented in Method 2, where detached silk segments were placed directly on water agar where they were inoculated with bacteria and Fg to promote dense colonization, and to allow for many replicates and interventions such as silk wounding. The bacterial endophytes were successfully observed colonizing Fg hyphae, silk trichomes, and entering silks via cut ends and wounds. These protocols can be used to study other silk-associated microbes including several globally important fungal pathogens that enter maize grain through silks.

Loss of Nkd1 is dominant over loss of Axin2 in regulating Wnt signaling

Bell, I. — 2023-11-02

Wnt signaling is a crucial developmental pathway involved in early development as well as stem cell maintenance in adults and its misregulation leads to numerous diseases. Thus, understanding the regulation of this pathway becomes vitally important. Axin2 and Nkd1 are widely utilized negative feedback regulators in Wnt signaling where Axin2 functions to destabilize cytoplasmic {beta}-catenin, and Nkd1 functions to inhibit the nuclear localization of {beta}-catenin. Here, we set out to further understand how Axin2 and Nkd1 regulate Wnt signaling by creating axin2-/-, nkd1-/- single mutants and axin2-/-;nkd1-/- double mutant zebrafish using sgRNA/Cas9. All three Wnt regulator mutants were viable and had impaired heart looping, neuromast migration defects, and behavior abnormalities in common, but there were no signs of synergy in the axin2-/-;nkd1-/- double mutants. Further, Wnt target gene expression by qRT-PCR, and RNA-seq analysis and protein expression by mass spectrometry demonstrated that the double axin2-/-;nkd1-/- mutant resembled the nkd1-/- phenotype demonstrating that Axin2 functions upstream of Nkd1 and that loss of Nkd1 is epistatic to the loss of Axin2. In support of this, the data further demonstrates that Axin2 uniquely alters the properties of {beta}-catenin-dependent transcription having novel readouts of Wnt activity compared to nkd1-/- or the axin2-/-;nkd1-/- double mutant. We also tested the sensitivity of the Wnt regulator mutants to exacerbated Wnt signaling, where the single mutants displayed characteristic heightened Wnt sensitivity, resulting in an eyeless phenotype. Surprisingly, this phenotype was rescued in the double mutant, where we speculate that cross-talk between Wnt/{beta}-catenin and Wnt/Planar Cell Polarity pathways could lead to altered Wnt signaling in some scenarios. Collectively, the data emphasizes both the commonality and the complexity in the feedback regulation of Wnt signaling.

Age-related blunting of serial sarcomerogenesis and mechanical adaptations following 2 weeks of maximal eccentric resistance training

Hinks, A. — 2023-11-09

BackgroundDuring natural aging, muscles atrophy, which is partly accounted for by a loss of sarcomeres in series. Serial sarcomere number (SSN) is associated with aspects of muscle mechanical function including the force-length and force-velocity-power relationships; hence, the age-related loss of SSN contributes to declining performance. Training emphasizing muscle lengthening (eccentric) contractions increases SSN in young healthy rodents. However, the ability for eccentric training to increase SSN and improve mechanical function in old age is unknown. MethodsTen young (9 months) and 11 old (33 months) Fisher344/BN F1 rats completed 4 weeks of unilateral isokinetic eccentric plantar flexion training 3 days/week. Pre- and post-training, the plantar flexors were assessed for maximum tetanic torque (ankle angles of 70{degrees} and 90{degrees}), the torque-frequency relationship (stimulation frequencies of 1-100 Hz), the passive torque-angle relationship (ankle angles of 110-70{degrees}), and the torque-angular velocity-power relationship (isotonic loads of 10%-80% maximum). Following post-training testing, rats were sacrificed, and the soleus, lateral gastrocnemius (LG), and medial gastrocnemius (MG) were harvested for SSN assessment by measuring sarcomere lengths with laser diffraction, with the untrained leg used as a control. ResultsIn the untrained leg/pre-training, old rats had lower SSN in the soleus (-9%), LG (-7%), and MG (-14%), lower maximum torque (-27 to -42%), power (-63%), and shortening velocity (-35%), and greater passive torque (+62 to +191%) than young. Young rats showed increased SSN from the untrained to the trained soleus and MG. In contrast, old rats had no change in soleus SSN between legs and experienced SSN loss in the LG. Pre- to post-training, young rats saw modest improvements in isometric mechanical function, including a 13% increase in maximum torque at 90{degrees} and 4-11% increases in 10-60 Hz torque. Old rats, however, had reductions in maximum torque (-35%), shortening velocity (-46%), and power (-63%), and increased passive torque (+24 to +51%) from pre- to post-training. ConclusionsEccentric training induced serial sarcomerogenesis and improved mechanical function in young rats, while old rats exhibited dysfunctional remodeling that led to impairments in muscle mechanical performance following training.

Latitudinal gradients in predation persist in urban environments

Hargreaves, A. L. — 2023-11-16

Urbanization can profoundly disrupt local ecology. But while urban areas now stretch across latitudes, little is known about urbanizations effects on macroecological patterns. We used standardized experiments to test whether urbanization disrupts latitudinal gradients in seed predation, a macroecological pattern that shapes community assembly and diversity. Using >56,000 seeds, we compared predation in urbanized and natural areas across 14,000 km of latitude, spanning the Americas. Predation increased 5-fold from high latitudes to the tropics, and latitudinal gradients in predation persisted in urban areas despite significant habitat modification. Urbanization reduced predation by vertebrates, but not invertebrates, and seemed to increase ant predation specifically. Our results show that macroecological patterns in predation intensity can persist in urbanized environments, even as urbanization alters the relative importance of predators. One-Sentence SummaryAcross 56,000 seeds and 112{degrees} of latitude, latitudinal gradients in seed predation are equally strong in natural vs. urban areas

Avian influenza viruses in wild birds in Canada following incursion of the highly pathogenic H5N1 virus from Eurasia in 2021/2022

Giacinti, J. A. — 2023-11-24

Following detection of novel highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b in Newfoundland, Canada in late 2021, avian influenza surveillance in wild birds was scaled-up across Canada. Herein, we present results of Canadas Interagency Surveillance Program for Avian Influenza in wild birds during the first year (November 2021 - November 2022) following the incursions of HPAIV from Eurasia. Key objectives of the surveillance program were to (i) detect the presence, distribution and spread of HPAIV and other avian influenza viruses (AIVs), (ii) detect wild bird morbidity and mortality associated with HPAIV, (iii) identify the range of wild bird species infected by HPAIV, and (iv) characterize detected AIV. A total of 6,246 sick and dead wild birds were tested, of which 27.4% were HPAIV positive across 12 taxonomic orders and 80 species. Geographically, HPAIV detections occurred in all Canadian provinces and territories, with the highest numbers in the Atlantic and Central flyways. Temporally, peak detections differed across flyways, though the national peak occurred in April 2022. In an additional 11,295 asymptomatic harvested or live captured wild birds, 5.2% were HPAIV positive across 3 taxonomic orders and 19 species. Whole genome sequencing identified HPAIV of Eurasian origin as most prevalent in the Atlantic flyway, along with multiple reassortants of mixed Eurasian and North American origins distributed across Canada, with moderate structuring at the flyway scale. Wild birds were victims and reservoirs of HPAIV H5N1 2.3.4.4b, underscoring the importance of surveillance encompassing samples from sick and dead, as well as live and harvested birds to provide insights into the dynamics and potential impacts of the HPAIV H5N1 outbreak. This dramatic shift in presence and distribution of HPAIV in wild birds in Canada highlights a need for sustained investment in wild bird surveillance and collaboration across One Health partners.

Urban-use pesticides in stormwater ponds and their accumulation in biofilms

Izma, G. — 2023-11-29

Stormwater ponds frequently receive urban runoff, increasing the likelihood of pesticide contamination. Biofilms growing in surface waters of these ponds are known to accumulate a range of aquatic contaminants, paradoxically providing both water purification services and potentially posing a threat to urban wildlife. Thus, sampling biofilms in stormwater ponds may be a critical and biologically relevant tool for characterizing pesticide contamination and toxicity in urban environments. Here, we aimed to investigate pesticide occurrences at 21 stormwater ponds in Brampton, ON, one of Canadas fastest growing municipalities, and quantify their accumulation in biofilm. Over nine weeks, we collected time-integrated composite water and biofilm samples for analysis of [~]500 current-use and legacy pesticides. Thirty-two pesticide compounds were detected across both matrices, with 2,4-D, MCPA, MCPP, azoxystrobin, bentazon, triclopyr, and diuron having near-ubiquitous occurrences. Several compounds not typically monitored in pesticide suites (e.g., melamine and nicotine) were also detected, but only in biofilms. Overall, 56% of analytes detected in biofilms were not found in water samples, indicating traditional pesticide monitoring practices fail to capture all exposure routes, as even when pesticides are below detection levels in water, organisms may still be exposed via dietary pathways. Calculated bioconcentration factors ranged from 4.2 - 1275 and were not predicted by standard pesticide physicochemical properties. Monitoring biofilms provides a sensitive and comprehensive supplement to water sampling for pesticide quantification in urban areas, and identifying pesticide occurrences in stormwater could improve source-tracking efforts in the future. Further research is needed to understand the mechanisms driving pesticide accumulation, to investigate toxicity risks associated with pesticide-contaminated biofilm, and to evaluate whether pesticide accumulation in stormwater pond biofilms represents a route through which contaminants are mobilized into the surrounding terrestrial and downstream aquatic environments.

Barcode 100K Specimens: In a Single Nanopore Run

Hebert, P. D. N. — 2023-11-30

It is a global priority to better manage the biosphere, but action needs to be informed by monitoring shifts in the abundance and distribution of species across the domains of life. The acquisition of such information is currently constrained by the limited knowledge of biodiversity. Among the 20 million or more species of eukaryotes, just a tenth have scientific names. DNA barcoding can speed the registration of unknown animal species, the most diverse kingdom of eukaryotes, as the BIN system automates their recognition. However, inexpensive analytical protocols are critical as the census of all animal species will require processing a billion or more specimens. Barcoding involves DNA extraction followed by PCR and sequencing with the last step dominating costs until 2017. By recovering barcodes from highly multiplexed samples, the Sequel platforms from Pacific BioSciences slashed costs by 90%, but these instruments are only deployed in core facilities because of their expense. Sequencers from Oxford Nanopore Technologies provide an escape from high capital and service costs, but their low sequence fidelity has, until now, kept analytical cost above Sequel. However, the improved performance of its latest flow cells (R10.4.1) might erase this differential. This study demonstrates that a regular MinION flow cell can characterize an amplicon pool derived from 100,000 specimens while a Flongle flow cell can process one derived from several thousand. At $0.01 per specimen, DNA sequencing is now the least expensive step in the barcode workflow. By coupling simplified protocols for DNA extraction with ultra-low volume PCRs, it will be possible to move from specimen to DNA barcode for $0.10, a price point that will enable the census of all species within two decades.

Enable, Empower, Succeed: Harnessing Open Science for Antimicrobial Resistance Containment

Founou, L. L. — 2024-02-23

Antimicrobial resistance (AMR) poses a significant threat to global health, particularly in Western sub-Saharan Africa where 27.3 deaths per 100,000 lives are affected, and surveillance and control measures are often limited. Genomics research plays a crucial role in understanding the emergence, spread and containment measures of AMR. However, its implementation in such settings is particularly challenging due to limited human capacity. This manuscript outlines a three-day bioinformatics workshop in Cameroon, highlighting efforts to build human capacity for genomics research to support AMR surveillance using readily accessible and user-friendly web-based tools. The workshop introduced participants to basic next-generation sequencing concepts, data file formats used in bacterial genomics, data sharing procedures and considerations, as well as the use of web-based bioinformatics software to analyse genomic data, including in silico prediction of AMR, phylogenetics analyses, and a quick introduction to Linux(C) command line. We provide a detailed description of the relevant training approaches used, including workshop structure, the selection and planning, and utilization of freely available web-based tools, and the evaluation methods employed. Our approach aimed to overcome limitations such as inadequate infrastructure, limited access to computational resources, and scarcity of expertise. By leveraging the power of freely available web-based tools, we demonstrated how participants can acquire fundamental bioinformatics skills, enhance their understanding of biological data analysis, and contribute to the field, even in an underprivileged environment. Our findings highlight the effectiveness of this training approach in empowering local researchers and bridging the bioinformatics gap in genomics surveillance of AMR in resource-constrained settings. Building human capacity for genomics research globally, and especially in resource-constrained settings, is imperative for ensuring global health and sustainable containment of AMR. Data summaryThe authors confirm that all supporting data, code and protocols have been provided within the article or through supplementary data files. Impact StatementAntimicrobial resistance (AMR) is a major global health threat, especially in Western sub-Saharan Africa, where 27.3 deaths per 100,000 lives occur. Genomics research play an instrumental role for understanding AMRs emergence, spread, and containment measures. However, its implementation in these settings is challenging due to limited human capacity. A three-day bioinformatics workshop in Cameroon aimed to build human capacity for genomics research using web-based tools. Participants were introduced to next-generation sequencing concepts, data file formats, data sharing procedures, and web-based bioinformatics software for analysing genomic data. The workshop aimed to overcome limitations like inadequate infrastructure, computational resources, and expertise scarcity. The findings show the effectiveness of this training approach in empowering local researchers and bridging the bioinformatics gap in genomics surveillance of AMR in resource-constrained settings.

Higher prevalence of sacbrood virus in highbush blueberry pollination units

McAfee, A. — 2024-03-22

Highbush blueberry pollination depends on managed honey bees (Apis mellifera) for adequate fruit set; however, beekeepers have raised concerns about poor health of colonies after pollinating this crop. Postulated causes include agrochemical exposure, nutritional deficits, and interactions with parasites and pathogens, particularly Melisococcus plutonius(the causal agent of European foulbrood disease), but other pathogens could be involved. To broadly investigate common honey bee pathogens in relation to blueberry pollination, we sampled adult honey bees from colonies at time points corresponding to before (t1), during (t2), at the end (t3), and after (t4) highbush blueberry pollination in British Columbia (BC), Canada, across two years (2020 and 2021). Nine viruses as well as M. plutonius, Vairimorpha ceranae and V. apis (formerly Nosema ceranae and N. apis) were detected by PCR and microscopy and compared among colonies located near and far from blueberry fields. We found a significant interactive effect of time and blueberry proximity on the multivariate pathogen community, mainly due to differences at t4 (corresponding to roughly six weeks after the beginning of the pollination period). Post-hoc comparisons of pathogens in near and far groups at t4 showed that detections of sacbrood virus (SBV), which was significantly higher in the exposed group, was the primary driver. The association of SBV with highbush blueberry pollination may be contributing to the health decline that beekeepers observe after pollinating this crop, likely in combination with other factors.

Intercontinental dissemination and diversification of Xanthomonas perforans in tomato production

Timilsina, S. — 2024-03-22

Emerging and re-emerging plant diseases continue to present multifarious threats to global food security. Considerable recent efforts are therefore being channeled towards understanding the nature of pathogen emergence, their spread and evolution. Xanthomonas euvesicatoria pv. perforans (Xep), one of the causal agents of bacterial spot of tomato, rapidly emerged and displaced other bacterial spot xanthomonads in tomato production regions around the world. In less than three decades, it has become a dominant xanthomonad pathogen in tomato production systems across the world and presents a model for understanding diversification of recently emerged bacterial plant pathogens. Although Xep has been continuously monitored in Florida since its discovery, the global population structure and evolution at the genome-scale is yet to be fully explored. The objectives of this work were to determine genetic diversity globally to ascertain if different tomato production regions contain genetically distinct Xep populations, to examine genetic relatedness of strains collected in tomato seed production areas in East Asia and other production regions, and to evaluate variation in type III effectors, which are critical pathogenicity and virulence factors, in relationship to population structure. We used genome data from 270 strains from 13 countries for phylogenetic analysis and characterization of Xop effector gene diversity among strains. Our results showed notable genetic diversity in the pathogen. We found genetically similar strains in distant tomato production regions, including seed production regions, and diversification over the past 100 years, which is consistent with intercontinental dissemination of the pathogen in hybrid tomato production chains. Evolution of the Xep pangenome, including the acquisition and loss of type III secreted effectors, is apparent within and among phylogenetic lineages. The apparent long-distance movement of the pathogen, together with variants that may not yet be widely distributed, poses risks of emergence of new variants in tomato production.

Building a Collaborative DNA Barcode Library for the Diptera of Churchill, Canada: A Resource for Northern Research

Majoros, S. E. — 2024-03-28

Churchill, Manitoba, Canada is a diverse Subarctic region that has been the focus of DNA barcoding efforts and research for decades. Despite this effort, there are still taxa in this region that are underrepresented in public databases, particularly hyperdiverse insect groups. We present a collaborative large-scale DNA barcode reference library for Diptera based on molecular data and expert taxonomic identification. The reference library contains 16786 specimens sampled around Churchill from 2005 to 2011. Specimens were identified by morphological means, sequenced for the animal barcode marker, and sorted into 2235 Molecular Operational Taxonomic Units (using Barcode Index Numbers: BINs) representing 68 families and 1211 named species. Eighty-four species shared a BIN with another species, 98 species were split across several BINs, and 1028 species were assigned to a unique BIN. The intraspecific and nearest neighbour distances varied across families, but most species can be easily distinguished from their nearest neighbours. By combining molecular data with taxonomic expertise, the barcode library provides species-level information for 12612 specimens, supplying future researchers with detailed taxonomic information and the opportunity to perform barcode-based specimen identifications for large-scale studies through sequence matching. Comparison of the Diptera composition of Churchill to other geographic regions reflected likely postglacial colonization northwards as well as a Beringian component. This study provides a publicly available, detailed reference library for use in future research, as well as new insights into the current diversity of Diptera present among the diverse habitats in this Subarctic region.

Nutrient addition in grasslands worldwide reveals proportional plant diversity decline across spatial scales but little change in beta diversity

Chen, Q. — 2024-03-29

Nutrient enrichment typically causes local plant diversity declines. A common but untested expectation is that nutrient enrichment also reduces variation in nutrient conditions among localities and selects for a smaller pool of species, causing greater diversity declines at larger than local scales and thus biotic homogenization. Here we apply a framework that links changes in species richness across scales to changes in the numbers of spatially restricted and widespread species for a standardized nutrient addition experiment across 72 grasslands on six continents. Overall, we find proportionally similar species loss at local and larger scales, suggesting similar declines of spatially restricted and widespread species, and no biotic homogenization after 4 years and up to 14 years of treatment. These patterns of diversity changes are generally consistent across species groups. Thus, nutrient enrichment poses threats to plant diversity, including for widespread species that are often critical for ecosystem functions.

Taxonomic Revision and Identification Keys for the Giant Honey Bees

Kitnya, N. — 2024-04-05

The taxonomy and phylogeny of the giant honey bees (Apis; subgenus Megapis) are still controversial and unresolved. The species boundaries within the subgenus are unclear and some species that are recognized on the basis of genetic differences lack supporting morphological characters. Two species are now well accepted: Apis dorsata F. of tropical regions of Asia and Apis laboriosa Smith that inhabits the foothills of Himalaya and neighboring mountain ranges. In addition, researchers have suggested that the two allopatric populations of giant honey bees that inhabit Sulawesi, Indonesia (A. binghami Cockerell) and the oceanic Philippine islands (A. breviligula Maa) as well as the South Indian form also deserve species status. We conducted a taxonomic study based on morphological characters of Megapis from throughout Asia. Our study confirms that Apis laboriosa is a distinct species based on numerous morphological characters. Moreover, A. dorsata of mainland Asia differs from the two island taxa based on coloration, ocellus size, and the spacing of compound eyes and ocelli. We found no evidence that breviligula of the Philippines has a distinctively short tongue, and report only one minor character (the shape of sternum 5) that differed statistically between bees from Sulawesi and the Philippines. We conclude that the bees from these islands represent a single morphological species, A. binghami, with two subspecies, A. b. binghami and A. b. breviligula. Apis dorsata from the Andaman Islands are smaller than but conspecific with those of mainland Asia. We found no morphological autapomorphies in the giant honey bees of southern India known to differ in mtDNA from A. dorsata from elsewhere in mainland Asia. We provide a taxonomic keys to workers and drones within the subgenus Megapis.

Dynamics of Saccade Trajectory Modulation by Distractors: Neural Activity Patterns in the Frontal Eye Field

Ramezanpour, H. — 2024-04-08

The sudden appearance of a visual distractor shortly before saccade initiation can capture spatial attention and modulate the saccade trajectory in spite of the ongoing execution of the initial plan to shift gaze straight to the saccade target. To elucidate the neural correlates underlying these curved saccades, we recorded from single neurons in the frontal eye field (FEF) of two rhesus monkeys shifting gaze to a target while an isoeccentric distractor appeared either left or right of the target at various delays after target presentation. We found that the population level of pre-saccadic activity encoded the direction of the saccade trajectory. Stronger activity occurred when saccades curved toward the distractor, and weaker when saccades curved away. This relationship held whether the distractor was ipsilateral or contralateral to the recorded neurons. Meanwhile, visually responsive neurons showed asymmetrical patterns of excitatory responses that varied with the location of the distractor and the duration of distractor processing relating to attentional capture and distractor inhibition. During earlier distractor processing, neurons encoded curvature towards the distractor. During later distractor processing, neurons encoded curvature away from the distractor. This was observed when saccades curved away from distractors contralateral to the recording site and when saccades curved towards distractors ipsilateral to the recording site. These findings indicate that saccadic motor planning involves dynamic push-pull hemispheric interactions producing attraction or repulsion for potential but unselected saccade targets. Significant StatementThis study not only advances our understanding of oculomotor function in dynamic environments but also has potential clinical relevance for diagnosing and understanding disorders characterized by abnormal saccade trajectories. Our research elucidates the neural mechanisms behind saccade trajectories that are not always linear due to the brains integration of multiple visual cues and/or motor plans. By analyzing the frontal eye field (FEF) activity in rhesus monkeys, we found that saccade directionality and timing are influenced by the interaction between FEF visual neurons representing target and distractor stimuli. The FEFs role extends beyond a winner-takes-all approach, incorporating saccade vector averaging computations that produce curved saccades. Furthermore, ipsilateral visual neurons encode distractor suppression that drives curvature away from the distractor.

The distribution of Apis laboriosa revisited: range extensions and biogeographic affinities

Otis, G. W. — 2024-04-07

Apis laboriosa, the Himalayan giant honeybee, inhabits the foothills of the Himalaya and neighboring mountainous regions. Here we revise its distribution in light of recent reports and discoveries. The range now extends from longitude 105.9{degrees}E in Cao Bang, Vietnam, in the east to 74.4{degrees}E in the Pir Panjal Range of western Himalaya, a linear distance of 3300 km, with the most notable new localities in northeastern Vietnam, central Myanmar, northern Thailand, and AJK-Pakistan. The species generally occurs at lower elevations in the eastern part of its range than in Nepal, northern India, and the border region between India and Pakistan. Most but not all of the new localities are within the range predicted by species distribution modelling. We discuss the new localities that fall outside of the predicted range, the biotic characteristics of the terrestrial ecoregions in which the species occurs, and the remaining regions that may harbor this spectacular honey bee species.

European Farmhouse Brewing Yeasts Form a Distinct Genetic Group

Preiss, R. — 2024-04-04

The brewing industry is constantly evolving, driven by the quest for novel flavors and fermentation characteristics that cater to evolving consumer preferences. This study explores the genetic and phenotypic diversity of European farmhouse yeasts, traditionally used in rural brewing practices and maintained outside of pure culture industrial yeast selection. We isolated landrace brewing yeast strains from diverse geographical locations across Europe, including Norway, Lithuania, Latvia, Russia, and also included African farmhouse brewing strains from Ghana. Our genomic analysis using long-read and short-read whole genome sequencing uncovered a genetically distinct group that diverges from industrial brewing yeasts. This group, which is closely related to ale brewing strains, is preliminarily named the "European Farmhouse" group and shows greater predicted admixture from Asian fermentation strains. Through genomic and phenotypic analyses, including flavor metabolite analysis via HS-GC-MS, sugar metabolite analysis via HPLC, and wort fermentation analysis, we found a broad spectrum of fermentation capabilities, from rapid and efficient fermentation to unique aroma and flavor compound profiles, potentially offering novel traits for brewing applications. This study highlights the importance of preservation of brewing cultural heritage knowledge and resources including yeast cultures. Key PointsO_LIA large set of geographically diverse farmhouse brewing strains were characterized C_LIO_LINorwegian and Baltic farmhouse brewing strains form a distinct genetic group C_LIO_LIFarmhouse strains show considerable diversity in fermentation and flavour formation C_LI

Muscle weakness and mitochondrial stress occur before metastasis in a novel mouse model of ovarian cancer cachexia

Delfinis, L. J. — 2024-04-08

ObjectivesA high proportion of women with advanced epithelial ovarian cancer (EOC) experience weakness and cachexia. This relationship is associated with increased morbidity and mortality. EOC is the most lethal gynecological cancer, yet no preclinical cachexia model has demonstrated the combined hallmark features of metastasis, ascites development, muscle loss and weakness in adult immunocompetent mice. MethodsHere, we evaluated a new model of ovarian cancer-induced cachexia with the advantages of inducing cancer in adult immunocompetent C57BL/6J mice through orthotopic injections of EOC cells in the ovarian bursa. We characterized the development of metastasis, ascites, muscle atrophy, muscle weakness, markers of inflammation, and mitochondrial stress in the tibialis anterior (TA) and diaphragm [~]45, [~]75 and [~]90 days after EOC injection. ResultsPrimary ovarian tumour sizes were progressively larger at each time point while robust metastasis, ascites development, and reductions in body, fat and muscle weights occurred by 90 Days. There were no changes in certain inflammatory (TNF), atrogene (MURF1 and Atrogin) or GDF15 markers within both muscles whereas IL-6 was increased at 45 and 90 Day groups in the diaphragm. TA weakness in 45 Day preceded atrophy and metastasis that were observed later (75 and 90 Day, respectively). The diaphragm demonstrated both weakness and atrophy in 45 Day. In both muscles, this pre-metastatic muscle weakness corresponded with considerable reprogramming of gene pathways related to mitochondrial bioenergetics as well as reduced functional measures of mitochondrial pyruvate oxidation and creatine-dependent ADP/ATP cycling as well as increased reactive oxygen species emission (hydrogen peroxide). Remarkably, muscle force per unit mass at 90 days was partially restored in the TA despite the presence of atrophy and metastasis. In contrast, the diaphragm demonstrated progressive weakness. At this advanced stage, mitochondrial pyruvate oxidation in both muscles exceeded control mice suggesting an apparent metabolic super-compensation corresponding with restored indices of creatine-dependent adenylate cycling. ConclusionThis mouse model demonstrates the concurrent development of cachexia and metastasis that occurs in women with EOC. The model provides physiologically relevant advantages of inducing tumour development within the ovarian bursa in immunocompetent adult mice. Moreover, the model reveals that muscle weakness in both TA and diaphragm precedes metastasis while weakness also precedes atrophy in the TA. An underlying mitochondrial bioenergetic stress corresponded with this early weakness. Collectively, these discoveries can direct new research towards the development of therapies that target pre-atrophy and pre-metastatic weakness during EOC in addition to therapies targeting cachexia. HighlightsO_LIThis study reports the first orthotopic model of metastatic ovarian cancer cachexia that can be induced in adult immunocompetent mice C_LIO_LIDiaphragm and limb muscle weakness precedes metastasis and atrophy during ovarian cancer C_LIO_LISkeletal muscle mitochondrial oxidative and redox stress signatures occur during pre-metastatic stages of ovarian cancer C_LIO_LISpecific muscle force as well as mitochondrial pyruvate oxidation and creatine metabolism demonstrate compensation in later stages C_LIO_LIOvarian cancer has heterogeneous effects on distinct muscle types across time C_LI

Transgenerational epigenetic inheritance increases trait variation but is not adaptive

Shahmohamadloo, R. S. — 2024-04-20

Understanding processes that can produce adaptive phenotypic shifts in response to rapid environmental change is critical to reducing biodiversity loss. The ubiquity of environmentally induced epigenetic marks has led to speculation that epigenetic inheritance could potentially enhance population persistence in response to environmental change. Yet, the magnitude and fitness consequences of epigenetic marks carried beyond maternal inheritance are largely unknown. Here, we tested how transgenerational epigenetic inheritance (TEI) shapes the phenotypic response of Daphnia clones to the environmental stressor Microcystis. We split individuals from each of eight genotypes into exposure and control treatments (F0 generation) and tracked the fitness of their descendants to the F3 generation. We found transgenerational epigenetic exposure to Microcystis led to reduced rates of survival and individual growth and no consistent effect on offspring production. Increase in trait variance in the F3 relative to F0 generations suggests potential for heritable bet hedging driven by TEI, which could impact population dynamics. Our findings are counter to the working hypothesis that TEI is a generally adaptive mechanism likely to prevent extinction for populations inhabiting rapidly changing environments. One sentence summaryTransgenerational epigenetic inheritance in Daphnia exposed to Microcystis revealed negative fitness effects on survival and growth rates, challenging hypotheses of a general selective advantage.

Yap is a Nutrient Sensor Sensitive to the Amino Acid L-Isoleucine and Regulates Expression of Ctgf in Cardiomyocytes.

Nelson, V. L. — 2024-04-28

Myocardial infarction and reperfusion is a complex injury consisting of many distinct molecular stress patterns that influence cardiomyocyte survival and adaptation. Cell signalling that is essential to cardiac development also presents potential disease-modifying opportunities to recover and limit myocardial injury or maladaptive remodelling. Here we hypothesized that Yap signalling could be sensitive to one or more molecular stress patterns associated with early acute ischemia. Yap, not Taz, patterns of expression differ in post-myocardial infarct compared to peri-infarct tissue suggesting cell-specificity that would be challenging to resolve for causation in vivo. Using H9c2 ventricular myotubes in vitro as a model, Yap levels were most sensitive to nutrient deprivation compared to other stress patterns typified by ischemia within the first hour of stress. Moreover, this is mediated by amino acid availability, dominantly L-isoleucine, and influences the expression of Ctgf--a major determinant of myocardial adaptation after injury. These findings present novel opportunities for future therapeutic development and risk assessment for myocardial injury and adaptation.

Engineered Ubiquitin Variants Mitigate Pathogenic Bacterial Ubiquitin Ligase Function

Dubrule, B. E. — 2024-05-01

During infection some pathogenic gram-negative bacteria, such as Salmonella, manipulate the host ubiquitination system through the delivery of secreted effectors known as novel E3 ubiquitin ligases (NELs). Despite the presence of NELs amongst these well-studied bacterial species, their unique structure has limited the tools that are available to probe their molecular mechanisms and explore their therapeutic potential. In this work, we report the identification of two high affinity engineered ubiquitin variants that can modulate the activity of the Salmonella enterica serovar Typhimurium encoded NEL, SspH1. We show that these ubiquitin variants suppress SspH1-mediated toxicity phenotypes in Saccharomyces cerevisiae. Additionally, we provide microscopic and flow cytometric evidence that SspH1-mediated toxicity is caused by interference with S. cerevisiae cell cycle progression that can be suppressed in the presence of ubiquitin variants. In vitro ubiquitination assays revealed that these ubiquitin variants increased the amount of SspH1-mediated ubiquitin chain formation. Interestingly, despite the increase in ubiquitin chains, we observe a relative decrease in the formation of SspH1-mediated K48-linked ubiquitin chains on its substrate, PKN1. Taken together our findings suggest that SspH1 toxicity in S. cerevisiae occurs through cell cycle interference and that an engineered ubiquitin variant approach can be used to identify modulators of bacterially encoded ubiquitin ligases. Author SummaryNovel E3 ligases (NELs) are a family of secreted effectors found in various pathogenic gram- negative bacteria. During infection these effectors hijack vital host ubiquitin signaling pathways to aid bacterial invasion and persistence. Despite interacting with a protein as highly conserved as ubiquitin, they have a distinct architecture relative to the eukaryotic E3 enzymes. This unique architecture combined with the indispensable role ubiquitin signaling plays in host cell survival has made hindering the contribution of NELs to bacterial infections a difficult task. Here, we applied protein engineering technology to identify two ubiquitin variants (Ubvs) with high affinity for SspH1, a Salmonella-encoded NEL. We provide evidence that these high affinity Ubvs suppress a known SspH1-meidated toxicity phenotype in the eukaryotic model system Saccharomyces cerevisiae. We also show that this suppression occurs without interfering with host ubiquitin signaling. Furthermore, we demonstrate the ability of a Ubv to modulate the activity of SspH1 in vitro, ultimately altering the lysine linkages found in SspH1-mediated ubiquitination. To our knowledge, this is the first evidence that an engineered ubiquitin variant approach can be implemented to modulate the activity of a family of previously untargetable bacterial-encoded E3 ligases.

HEPN-AbiV is an RNase in the antiphage system AbiV

Zhu, X. — 2024-05-05

Prokaryotes and eukaryotes possess defense systems, which can be either innate or acquired, to protect against viral infections. At the bacterial population level, abortive infection (Abi) serves as an innate immune defense mechanism against phage invasion. The AbiV antiviral system is prevalent in several bacterial genomes and exhibits diverse characteristics in terms of gene composition and evolution. Our investigation into the Lactococcus AbiV system revealed a novel two-component system, abiV1 and abiV2, both of which are essential for its function as a type III toxin-antitoxin system. The toxin component AbiV (product of abiV1) is an RNase belonging to the HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) superfamily as it carries the consensus Rx4-6H motif. In vivo assays coupled with mass spectrometry showed that the lactococcal AbiV was expressed in the presence or absence of phages while in vitro experiments demonstrated that AbiV1 degraded ribosomal RNA but not mRNA. On the other hand, the antitoxin component (abiV2) was found to function as an RNA molecule that inhibited the nuclease activity of the AbiV1 toxin. The structural characterization of AbiV revealed that this RNase utilizes a large patch of positively charged area across the dimer to anchor RNA molecules. In addition, we showed that the AbiV N-terminal region (amino acids 1 to 23) is crucial for its RNase activity as a truncated AbiV lacking this segment adopted distinct conformational states incompatible with RNA binding. This study provided novel insights into the mode of action of the antiviral system AbiV.

Phylogenomics of novel clones of Aeromonas veronii recovered from a freshwater lake reveals unique biosynthetic gene clusters

Lawal, O. U. — 2024-05-10

Aquatic ecosystems are important reservoirs for clinically relevant pathogens and antimicrobial resistance genes, thus present a significant risk to global health. Here, we assessed the phylogenomics of Aeromonas veronii (A. veronii) recovered from Lake Wilcox in Ontario using a combination of morphological, biochemical, and whole-genome sequencing (WGS) techniques. Eleven distinct bacterial colonies were isolated and identified as A. veronii (n=9), and two other Aeromonas species (A. caviae and A. allosaccharophila), with significant discrepancies noted between biochemical and WGS identification methods. Of note, 67% (n=6/9) of A. veronii isolates were human pathogens (Pathogenicity score [≥] 0.50). The genomic analysis revealed high genetic diversity among the A. veronii isolates, including the discovery of 41 novel alleles and seven new sequence types (ST) suggesting the lake as a reservoir for multiple human pathogenic clones of this bacterium. The comparison of the newly isolated and sequenced A. veronii with 214 A. veronii genomes revealed significant genetic diversity and suggests potential broad geographical dissemination of strains. Chromosomal genes (OXA-912 and cphA [cphA3, cphA4, cphA7]) genes encoding resistance to {beta}-lactamases were detected in all isolates. Human and non-human pathogenic strains of A. veronii differed in their virulence gene content, with type III secretion systems being associated with human pathogenic isolates. Mobilome analysis revealed the absence of plasmids in A. veronii isolates and the presence of 13 intact the great majority of which were P22-like (Peduoviridae) phages, and nine different insertion sequence families. Novel biosynthetic gene clusters were identified and characterized, indicating the potential for unique secondary metabolite production in A. veronii with different pathogenic potential. Overall, this study underscores the importance of continuous surveillance of aquatic ecosystems for the presence of pathogens, contributing to our understanding of their evolution, potential for human pathogenicity, and the ecological roles of their genetic elements.

11β-hydroxysteroid dehydrogenase type 2 may mediate the stress-specific effects of cortisol on brain cell proliferation in adult zebrafish (Danio rerio)

Flatt, E. E. — 2024-05-17

Stress-induced increases in cortisol can stimulate or inhibit brain cell proliferation, but the mechanisms behind these opposing effects are unknown. We tested the hypothesis that 11{beta}-hydroxysteroid dehydrogenase type 2 (Hsd11b2), a glucocorticoid-inactivating enzyme expressed in neurogenic regions of the adult zebrafish brain, mitigates cortisol-induced changes to brain cell proliferation using one of three stress regimes: a single 1-min air exposure (acute stress), two air exposures spaced 24 h apart (repeat acute stress), or social subordination (chronic stress). Plasma cortisol was significantly elevated 15 min after air exposure and recovered within 24 h after acute and repeat stress, whereas subordinate fish exhibited significant and sustained elevations relative to dominant fish for 24 h. Following acute stress, brain hsd11b2 transcript abundance was significantly lower 24 h after a single air exposure but was unchanged by repeat stress or social subordination. A sustained increase in brain Hsd11b2 protein levels occurred after acute stress, but not after repeat or chronic stress. Following acute and repeat stress, brain pcna transcript abundance exhibited a prolonged elevation, but was unaffected by social subordination. Interestingly, the number of telencephalic BrdU+ cells increased in fish after a single air exposure but was unchanged by repeat stress. Following acute and repeat stress, fish expressed lower brain gr and mr transcript abundance while subordinate fish exhibited no changes. Taken together, these results demonstrate stressor-specific regulation of Hsd11b2 in the zebrafish brain that could modulate rates of cortisol catabolism contributing to observed differences in brain cell proliferation. Summary StatementThe steroid dehydrogenase, Hsd11b2, is dynamically regulated in the zebrafish brain during stress. This study provides novel evidence that variation in Hsd11b2 abundance may underscore stress-specific changes in forebrain neurogenesis.

Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes

Ramarapu, R. — 2024-05-21

IntroductionThe domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing. MethodsPeripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, alpha/beta T cell receptor transcripts and gamma/delta T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5 reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs. ResultsUnsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and gamma/delta T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis demonstrated a skewed T-cell receptor alpha gene usage and a restricted T-cell receptor gamma junctional length in CD8+ cytotoxic T cells compared to other alpha/beta T cell subsets. Among myeloid cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster. DiscussionOur study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

Effects of lysine and methionine on mRNA expression of candidate transcription factors by primary bovine mammary epithelial cells

Li, B. — 2024-06-01

It has been established that essential amino acids (EAA) regulate protein synthesis in mammary epithelial cells by rapidly altering the phosphorylation state of translation factors. However, the long-term transcriptional response to EAA supply has been investigated much less. Eight transcription factors were selected as candidate mediators of EAA effects on mammary cell function via the amino acid response (ATF4, ATF6), mitogen-activated protein kinase (JUN, FOS, EGR1), and mechanistic target of rapamycin complex 1 (MYC, HIF1A, SREBF1). The objective was to determine if and when expression of these candidate genes was affected in primary cultures of bovine mammary epithelial cells more than 24 h after imposing an EAA deficiency, and to evaluate effects of EAA deficiency on protein synthesis, endoplasmic reticulum size, cell proliferation, and lipogenesis. Differentiated cells were cultured in 1 of 3 treatment media representing normal physiological concentrations of all amino acids (CTL), low lysine (LK), or low methionine (LM) for 24, 40, 48, or 60 h. Both LK and LM suppressed protein synthesis and activated ATF4 expression, indicating the classic amino acid response pathway had been triggered. However, there was no effect of LK or LM on endoplasmic reticulum size, possibly related to elevated ATF6 expression on LM. Expression of early response genes JUN, FOS, EGR1 and MYC was not elevated by EAA deficiency but LM decreased EGR1 expression. LM also increased expression of HIF1A. The EGR1 and HIF1A expression results are consistent with the decrease in cell proliferation rate observed. Variable responses in SREBF1 expression to LK and LM at different timepoints may have contributed to a lack of effect on lipogenesis rates. These findings indicate that EAA deficiency may inhibit mammary protein synthesis and cell proliferation through transcription factors.

Reactivation-dependent transfer of fear memory between contexts requires M1 muscarinic receptor stimulation in dorsal hippocampus

Abouelnaga, K. H. — 2024-06-03

BackgroundMemory updating is essential for integrating new information into existing representations. However, this process could become maladaptive in conditions like post-traumatic stress disorder (PTSD), when fear memories generalize to neutral contexts. Previously, we have shown that contextual fear memory malleability in rats requires activation of M1 muscarinic acetylcholine receptors in the dorsal hippocampus. Here, we investigated the involvement of this mechanism in transfer of contextual fear memories to other contexts using a novel fear memory updating paradigm. MethodsFollowing brief re-exposure to a previously fear conditioned context, male rats (n=8-10/group) were placed into a neutral context to evaluate the transfer of fear memory. We also infused the selective M1 receptor antagonist pirenzepine into the dorsal hippocampus prior to memory reactivation to try to block this effect. ResultsResults support the hypothesis that fear memory can be updated with novel contextual information, but only if rats are re-exposed to the originally trained context relatively recently prior to the neutral context; evidence for transfer was not seen if the fear memory reactivation was omitted or if it occurred 6h prior to neutral context exposure. The transferred fear persisted for four weeks, and the effect was blocked by M1 antagonism. ConclusionsThese findings strongly suggest that fear transfer requires reactivation and destabilization of the original fear memory. Specific parameters likely dictate similar generalization in disorders like PTSD. The novel preclinical model introduced here, and its implication of muscarinic receptors in this process, should inform therapeutic strategies in this area.

Age-related differences in the loss and recovery of serial sarcomere number following disuse atrophy in rats

Hinks, A. — 2024-06-11

BackgroundOlder adults exhibit a slower recovery of muscle mass following disuse atrophy than young adults. At a smaller scale, muscle fibre cross-sectional area (i.e., sarcomeres in parallel) exhibits this same pattern. Less is known, however, about age-related differences in the recovery of muscle fibre length, driven by increases in serial sarcomere number (SSN), following disuse. The purpose of this study was to investigate age-related differences in SSN adaptations and muscle mechanical function during and following muscle immobilization. We hypothesized that old rats would experience a similar magnitude of SSN loss during immobilization, however, take longer to recover SSN than young following cast removal, which would limit the recovery of muscle mechanical function. MethodsWe casted the plantar flexors of young (8 months) and old (32 months) male rats in a shortened position for 2 weeks, and assessed recovery during 4 weeks of voluntary ambulation. Following sacrifice, legs were fixed in formalin for measurement of soleus wet weight and SSN with the un-casted soleus acting as a control. Ultrasonographic measurements of pennation angle (PA) and muscle thickness (MT) were also conducted weekly. In-vivo active and passive torque-angle relationships were constructed pre-cast, post-cast, and following 4 weeks of recovery. ResultsFrom pre- to post-cast, young and old rats experienced similar decreases in SSN (-20%, P<0.001), muscle wet weight (-25%, P<0.001), MT (-30%), PA (-15%, P<0.001), and maximum isometric torque (-40%, P<0.001), but there was a greater increase in passive torque in old (+180%, P<0.001) compared to young rats (+68%, P=0.006). Following cast removal, young exhibited quicker recovery of SSN, PA, and MT than old, but SSN recovered sooner than PA and MT in both young and old. Muscle wet weight recovered 90% and active torque fully recovered in young rats, whereas in old these remained unrecovered at 75% and 72%, respectively. ConclusionsThis study showed that old rats retain a better ability to recover longitudinal compared to parallel muscle morphology following cast removal, making SSN a highly adaptable, appealing mechanism for restoration of functional capacity following disuse in elderly populations.

Residual force enhancement decreases when scaling from the single muscle fibre to joint level in humans

Hinks, A. — 2024-06-11

Residual force enhancement (rFE), defined as increased isometric force following active lengthening compared to a fixed-end isometric contraction at the same muscle length and level of activation, is present across all scales of muscle. While rFE is always present at the cellular level, often rFE non-responders are observed during joint-level voluntary contractions. We compared rFE between the joint level and single fibre level (vastus lateralis biopsies) in 16 young males. In-vivo voluntary knee-extensor rFE was measured by comparing steady-state isometric torque between a stretch-hold (maximal activation at 150{degrees}, stretch to 70{degrees}, hold) and a fixed-end isometric contraction, with ultrasonographic recording of vastus lateralis fascicle length (FL). Fixed-end contractions were performed at 67.5{degrees}, 70{degrees}, 72.5{degrees}, and 75{degrees}; the joint angle that most closely matched FL of the stretch-hold contractions isometric steady-state was used to calculate rFE. The starting and ending FLs of the stretch-hold contraction were expressed as % optimal FL, determined via torque-angle relationship. In single fibre experiments, the starting and ending fibre lengths were matched relative to optimal length determined from in-vivo testing, yielding an average sarcomere excursion of [~]2.2-3.4{micro}m. There was a greater magnitude of rFE at the single fibre ([~]20%) than joint level ([~]5%) (P=0.004), with non-responders only observed at the joint level. By comparing rFE across scales within the same participants, we show the development of the rFE non-responder phenomenon is upstream of rFEs cellular mechanisms, with rFE only lost rather than gained when scaling from single fibres to the joint level.

No effect of menstrual cycle phase on eccentric exercise-induced neuromuscular impairments and the magnitude of the repeated bout effect

Rilling, A. — 2024-06-11

Neuromuscular function is impaired following an unaccustomed bout of eccentric exercise, however, through the repeated bout effect (RBE) the muscle is protected from damage following a subsequent bout of eccentric exercise. As a result of unaccustomed eccentric contractions, structural muscle damage occurs in both sexes. However, the inflammatory response may be mitigated in females due to estradiol, thereby attenuating the secondary effects of muscle damage and potentially limiting the magnitude of the RBE. We investigated the relationship between menstrual cycle phase and oral contraceptive use on neuromuscular impairments following the first bout of exercise, and the magnitude of the RBE. Thirteen female participants performed two bouts of 150 maximal eccentric voluntary contractions of the elbow flexors four weeks apart. Normally menstruating females participated during the late follicular phase (day 10-14) of their menstrual cycle, as determined through cycle tracking, when estradiol is near peak, and progesterone is lower. Oral contraceptive users were tested on their placebo pill days (lower estradiol). Neuromuscular function was assessed for Bout 1 before the eccentric protocol and then again 48 hours following, and this was repeated 4 weeks later for Bout 2. Eccentric exercise-induced muscle weakness and soreness did not differ between groups following Bout 1 (p=0.885), and the magnitude of the RBE (p<0.05) was similar between groups (p=0.995). Females in the late follicular phase (classified as high estradiol) and females on combined oral contraceptives (low estradiol) had similar impairments in neuromuscular function following the first bout of eccentric exercise, and a similar RBE.

Residual force enhancement is not altered while force depression is amplified at the cellular level in old age

Njai, B. S. — 2024-06-17

Residual force enhancement (rFE) and residual force depression (rFD) are history-dependent properties of muscle which refer to increased and decreased isometric force following a lengthening or shortening contraction, respectively. The history-dependence of force is greater in older than younger adults when assessed at the joint level. However, it is unclear whether this amplification of the history-dependence of force in old age is owing to cellular mechanisms or a consequence of age-related remodeling of muscle architecture. Single muscle fibres from the psoas major of old and young F344BN rats were dissected and chemically permeabilized. Single muscle fibres were mounted between a force transducer and length controller, then maximally activated (pCa 4.5). To assess rFD, fibers were actively shortened from 3.1 to 2.5{micro}m at both a slow (0.15Lo/s) and fast (0.6Lo/s) speed, with a fixed-end isometric reference contraction at 2.5{micro}m. To assess rFE, fibers were activated and stretched at 0.3Lo/s from a sarcomere length of 2.2 to 2.5{micro}m, and 2.7 to 3.0{micro}m, and compared to fixed-end isometric reference contractions at 2.5 and 3.0{micro}m, respectively. Isometric force was {approx}19% lower in old as compared with young (p<0.001). Upon normalizing to fibre cross-sectional area, there was no age-related difference in specific force (p>0.05). rFD was {approx}33% greater in old as compared with young (p<0.05), while rFE did not differ between groups (p>0.05). rFD is amplified in old age at the cellular level, while rFE appears to be unchanged, thus previously reported age-related modification of rFE occurs upstream from the cellular level.

Density-dependent network structuring within and across wild animal systems

Albery, G. F. — 2024-07-02

High population density should drive individuals to more frequently share space and interact, producing better-connected spatial and social networks [1-4]. Although this theory is fundamental to our understanding of disease dynamics [2,5-8], it remains unconfirmed how local density generally drives individuals positions within their networks, which reduces our ability to understand and predict density-dependent processes [4,9,10]. Here we provide the first general evidence that density drives greater network connectedness at fine spatiotemporal scales, at the scale of individuals within wild animal populations. We analysed 36 datasets of simultaneous spatial and social behaviour in >58,000 individual animals, spanning 30 species of fish, reptiles, birds, mammals, and insects. 80% of systems exhibited strong positive relationships between local density and network centrality. However, >80% of relationships were nonlinear and 75% became shallower at higher values, signifying that demographic and behavioural processes counteract densitys effects, thereby producing saturating trends [11-15]. Densitys effect was much stronger and less saturating for spatial than social networks, such that individuals become disproportionately spatially connected rather than socially at higher densities. Consequently, ecological processes that depend on spatial connections (e.g. indirect pathogen transmission, resource competition, and territory formation) are likely more density-dependent than those involving social interactions (e.g. direct pathogen transmission, aggression, and social learning). These findings reveal fundamental ecological rules governing societal structuring, with widespread implications. Identifying scaling rules based on processes that generalise across systems, such as these patterns of density dependence, might provide the ability to predict network structures in novel systems.

Submaximal eccentric resistance training increases serial sarcomere number and improves dynamic muscle performance in old rats

Hinks, A. A. — 2024-07-16

The age-related loss of muscle mass is partly accounted for by the loss of sarcomeres in series. Serial sarcomere number (SSN) influences muscle mechanical function including the force-length and force-velocity-power relationships, and the age-related loss of SSN contributes to declining performance. Resistance training biased to active lengthening (eccentric) contractions increases SSN in young muscle, however, we showed maximal eccentric training in old rats did not alter SSN and further worsened performance. A submaximal eccentric training stimulus may be more conducive to adaptation for aged muscle. Therefore, the purpose of this study was to assess whether submaximal eccentric training can increase SSN and improve mechanical function in old rats. Twelve 32-month-old male F344/BN rats completed 4 weeks of submaximal (60% maximum) isokinetic eccentric plantar-flexion training 3 days/week. Pre- and post-training, we assessed in-vivo maximum isometric torque at a stretched and neutral ankle angle, the passive torque-angle relationship, and the isotonic torque-angular velocity-power relationship. The soleus and MG were harvested for SSN measurements via laser diffraction, with the untrained leg as a control. SSN increased 11% and 8%, and muscle wet weight increased 14% and 13% in the soleus and MG, respectively. Maximum isometric torque and shortening velocity were unaltered, but there was a shift towards longer muscle lengths for the optimal angle of torque production, a 42% reduction of passive torque, and 23% increase in peak isotonic power. Eccentric training at 60% maximum was beneficial for aged muscle, increasing SSN and muscle mass, reducing muscle passive tension, and improving dynamic performance. New & NoteworthyO_LIFour weeks of submaximal (60% of maximum) eccentric resistance training increased serial sarcomere number and muscle mass in old rats C_LIO_LIIncreased serial sarcomere number corresponded to broadening of the torque-angle relationship following training, which contributed to increased peak isotonic power C_LIO_LISubmaximal eccentric training also led to a large reduction in passive torque throughout the range of motion C_LI

Welcome to Hotel Hymenoptera: monitoring cavity-nesting bee and wasp distribution and their trophic interactions using community science and metabarcoding

Handler, S. — 2024-07-16

Essential ecosystem services are provided by many interactions, including plant-pollinator, predator-prey, and host-parasitoid. These services support food and natural systems through pollination and pest control, however they are challenging to qualify, and previous observational studies may underestimate their complexity. The cavity nesting Hymenoptera are a good example showing all these three interactions and they can be monitored using trap nests. For this study, trap nests were installed at schools across Canada by community scientists to investigate cavity-nesting bee and wasp distributions and interactions. DNA metabarcoding was used to identify the occupants and their food sources. New bee and wasp distributions were found that might be the result of previous under-sampling or recent range expansions. Detailed bipartite and tripartite networks describing species interactions suggest some novel bee, wasp, and parasite associations. These results encourage further investigation into these interactions using molecular methods as detailed range maps and networks provide information to natural historians and conservationists alike.

Test of a conservation intervention highlights temporal variability in hybridization dynamics in Catostomus fishes

Campbell, J. N. — 2024-07-23

Non-native species are a leading threat to fish biodiversity. They pose risks to native populations through human-mediated introductions resulting in hybridization events, which could result in demographic or genetic swamping. Catostomus fishes in the Upper Colorado River Basin are an example of this. Extensive hybridization occurs between non-native white suckers (C. commersonii ) and native flannelmouth and bluehead suckers (C. latipinnis and C. discobolus). This system provides a suitable model for using genomic analyses to test the efficacy of an intervention to reduce the abundance of non-native species and production of hybrid offspring. This study implemented a Resistance Board Weir (RBW) as a fish barrier across Roubideau Creek, a tributary of the Gunnison River in Colorado (USA), to restrict non-native sucker participation in spawning events. Conducted over four years, the study gathered genomic data from larval fish samples, pre- and post-implementation of the RBW. We used genomic data to determine the efficacy of a RBW at limiting non-native and hybridized sucker larval production. We found no significant effect of the weir on the proportion of white sucker ancestry in larval fish across the four years of the study, which included three years of weir usage when access was successfully controlled for variable amounts of time. Overall, this work provides insight into the efficacy of a resistance board weir as a management tool for non-native suckers, and highlights interannual variability. This work contributes valuable information for policy and fisheries management in Colorado.

Investigating the temporal effects of ovarian failure on single muscle fibre contractility using a chemically-induced ovarian failure model in mice

Mashouri, P. — 2024-07-23

We investigated the effects of chemically-induced ovarian failure on single muscle fibre contractility of the soleus and extensor digitorum longus (EDL) muscles throughout ovarian failure, thereby mimicking the menopausal transition into late-stage menopause: [(D60;peri-menopause), (D120;onset of menopause), (D134;early-onset menopause), (D176;late-stage menopause)]. We used 4-vinylcyclohexene diepoxide (VCD) to induce ovarian failure in sexually-mature female mice. At D120 and D176, mice with VCD-induced ovarian failure produced higher force as compared with controls (p<0.05). On D134, however, VCD had lower force production compared with controls (p<0.05). The cross-sectional area of the soleus fibres from the VCD group was larger at D120 compared with controls (p<0.05), but not at any other time point (p>0.05). As well, at D120-D176, the proportion of Type II fibres relative to Type I increased for the soleus, but not the EDL. No differences in rate of force redevelopment (Ktr) was observed for the soleus (p>0.05), while calcium sensitivity increased by late-stage menopause (p<0.05). There were no differences in force, cross-sectional area, stiffness, Ktr, or calcium sensitivity between groups for the EDL (p>0.05). Muscle contractility across the peri-menopausal transition into late-stage menopause is both muscle and phase-dependent, emphasizing the complexity of changing hormones throughout the lifespan on muscle contractile function.

A leaf-expressed TERMINAL FLOWER1 ortholog from coffee with alternate splice forms alters flowering time and inflorescence branching in Arabidopsis

Cardon, C. — 2024-08-15

Perennial, polycarpic species, such as Coffea sp L. (coffee), exhibit asynchronous flowering while maintaining concomitant vegetative growth. This growth dichotomy is associated directly with fruit development and maturation time. To identify molecular components that underlie asynchronous flowering, we isolated phosphatidylethanolamine binding protein (PEBP) homologs expressed in coffee and identified a gene with high similarity to Arabidopsis TERMINAL FLOWER1-like. In Arabidopsis, interaction of TFL1 (AtTFL1) with bZIP transcription factor floral regulator FD (AtFD) forms a floral repressor complex that maintains inflorescence meristems in an indeterminate state. Unlike AtTFL1, which is expressed only in the shoot apical meristem, CaTFL1 transcript was detected exclusively in coffee leaves. Moreover, this transcript retained an intron, which was not reported for AtTFL1. CaTFL1 was characterized through heterologous expression in Arabidopsis and protein interaction analysis. Ectopic overexpression of CaTFL1 in transgenic Arabidopsis plants caused extreme late flowering or prevented flowering. However, the most severe floral repressive activity occurred in transgenic plants that spliced out the extra intron from CaTFL1. Yeast two hybrid assay revealed that CaTFL1 protein encoded by the spliced mRNA interacts with AtFD, as well as Arabidopsis 14-3-3 protein. These findings suggest that CaTFL1 acts as a leaf-expressed floral repressor, whose activity is controlled by alternate splicing, and may contribute to asynchronous flowering in coffee.

A test for microbiome-mediated rescue via host phenotypic plasticity in Daphnia

Shahmohamadloo, R. S. — 2024-08-15

Phenotypic plasticity is a primary mechanism by which organismal phenotypes shift in response to the environment. Host-associated microbiomes often exhibit considerable shifts in response to environmental variation and these shifts could facilitate host phenotypic plasticity, adaptation, or rescue populations from extinction. However, it is unclear how much shifts in microbiome composition contribute to host phenotypic plasticity, limiting our knowledge of the underlying mechanisms of plasticity and, ultimately, the fate of populations inhabiting changing environments. In this study, we examined phenotypic responses and microbiome composition in 20 genetically distinct Daphnia magna clones exposed to non-toxic and toxic diets containing Microcystis, a cosmopolitan cyanobacteria and common stressor for Daphnia. Daphnia exhibited significant plasticity in survival, reproduction, and population growth rates in response to Microcystis exposure. However, the effects of Microcystis exposure on the Daphnia microbiome were limited, with the primary effect being differences in abundance observed across five bacterial families. Moreover, there was no significant correlation between the magnitude of microbiome shifts and host phenotypic plasticity. Our results suggest that microbiome composition played a negligible role in driving host phenotypic plasticity or microbiome-mediated rescue. One sentence summaryDaphnia exhibits considerable plasticity in individual and population-level responses to a cosmopolitan stressor, yet shifts in microbiome composition are not correlated with the magnitude of this plasticity.

Pollen foraging mediates exposure to dichotomous stressor syndromes in honey bees

Wizenberg, S. B. — 2024-08-20

Recent declines in the health of honey bee colonies used for crop pollination pose a considerable threat to global food security. Foraging by honey bee workers represents the primary route of exposure to a plethora of toxins and pathogens known to affect bee health, but it remains unclear how foraging preferences impact colony-level patterns of stressor exposure. Resolving this knowledge gap is crucial for enhancing the health of honey bees and the agricultural systems that rely on them for pollination. To address this, we carried out a national-scale experiment encompassing 456 Canadian honey bee colonies to first characterize pollen foraging preferences in relation to major crops, then explore how foraging behaviour influences patterns of stressor exposure. We used a metagenetic approach to quantify honey bee dietary breadth and found that bees display distinct foraging preferences that vary substantially relative to crop type and proximity, and the breadth of foraging interactions can be used to predict the abundance and diversity of stressors a colony is exposed to. Foraging on diverse plant communities was associated with increased exposure to pathogens, while the opposite was associated with increased exposure to xenobiotics. Our work provides the first large-scale empirical evidence that pollen foraging behaviour plays an influential role in determining exposure to dichotomous stressor syndromes in honey bees. Significance StatementInsect-mediated pollination is an important ecological process that is crucial for food production. Managed honey bee colonies are one of the most important insect pollinators, but their health has been under threat from a variety of stressors. Bee workers are primarily exposed to stressors while foraging and understanding how bee foraging preferences are related to exposure risk could provide pivotal information to improve management efforts. Here, we studied honey bee foraging preferences in relation to prominent Canadian crops and across a gradient of modified environments. We found that honey bees show distinct, measurable foraging preferences and that dietary diversity is a strong predictor of the stressors that colonies are exposed to.

Common to rare transfer learning (CORAL) enables inference and prediction for a quarter million rare Malagasy arthropods

Ovaskainen, O. — 2024-08-22

Modern DNA-based biodiversity surveys result in massive-scale data, including up to millions of species - of which most are rare. Making the most of such data for inference and prediction requires modelling approaches that can relate species occurrences to environmental and spatial predictors, while incorporating information about their taxonomic or phylogenetic placement. Even if the scalability of joint species distribution models to large communities has greatly advanced, incorporating hundreds of thousands of species has not been feasible to date, leading to compromised analyses. Here we present a novel "common to rare transfer learning" approach (CORAL), based on borrowing information from the common species to enable statistically and computationally efficient modelling of both common and rare species. We illustrate that CORAL leads to much improved prediction and inference in the context of DNA metabarcoding data from Madagascar, comprising 255,188 arthropod species detected in 2874 samples.

Prenatal exposure to valproic acid induces sex-specific alterations in cortical and hippocampal neuronal structure and function in rats

Williams, O. O. F. — 2024-09-05

BackgroundThere are substantial differences in the characteristics of males and females with an autism spectrum disorder (ASD), yet there is little knowledge surrounding the mechanistic underpinnings of these differences. The valproic acid (VPA) rodent model is the most widely used model for the study of idiopathic ASD, but almost all of the studies have used male rodents. MethodTo fill this knowledge gap, we evaluated sex differences for neuronal activity, morphology, and glycogen synthase kinase-3 (GSK-3) signaling in primary cortical (CTX) and hippocampal (HIP) neurons prepared from rats exposed to VPA in utero. In vivo, sex-specific VPA-induced alterations in the frontal CTX transcriptome at birth were also determined. ResultsOverall, VPA induced more robust changes in neuronal function and structure in the CTX than in the HIP. Male- and female-derived primary CTX neurons from rats exposed to prenatal VPA had elevated activity and showed more disorganized firing. In the HIP, only the female VPA neurons showed elevated firing, while the male VPA neurons exhibited disorganized activity. Dendritic arborization of CTX neurons from VPA rats was less complex in both sexes, though this was more pronounced in the females. Conversely, both female and male HIP neurons from VPA rats showed elevated complexity distal to the soma. Female VPA CTX neurons also had an elevated number of dendritic spines. The relative activity of the and {beta} isoforms of GSK-3 were suppressed in both female and male VPA CTX neurons, with no changes in the HIP neurons. On postnatal day 0, alterations in CTX genes associated with neuropeptides (e.g., penk, pdyn) and receptors (e.g., drd1, adora2a) were seen in both sexes, though they were downregulated in females and upregulated in males. LimitationsPrimary neuron studies may not recapitulate findings performed in vivo or at later stages of development. ConclusionTogether these findings suggest that substantial sex differences in neuronal structure and function in the VPA model may have relevance to the reported sex differences in idiopathic ASD.

Genomic characterization of foodborne Salmonella enterica and Escherichia coli isolates from Saboba district and Bolgatanga Municipality Ghana

Sunmonu, G. T. — 2024-09-06

Salmonella enterica and Escherichia coli are well-known bacteria commonly associated with foodborne illnesses in humans and animals. Genomic characterization of these pathogens provides valuable insights into their evolution, virulence factors and resistance determinants. This study aimed to characterized previously isolated Salmonella (n = 14) and E. coli (n = 19) from milk, meat and its associated utensils in Ghana using whole-genome sequencing. Most of the Salmonella serovars (Fresno, Plymouth, Infantis, Give and Orleans) identified in this study are yet to be reported in Ghana. Most Salmonella isolates were pan-sensitive, but genes conferring resistance to fosfomycin (fosA7.2) and tetracycline (tet(A)) were detected in one and three isolates, respectively. Seven of the Salmonella isolates carry the IncI1-I(Gamma) plasmid replicon. Although antimicrobial resistance was not common among Salmonella strains, most (11/19) of the E. coli strains had at least one resistance gene, with nearly half (8/19) being multidrug resistant and carrying mobile elements. Three of the 19 E. coli strains belong to serovars commonly associated with enteroaggregative E. coli (EAEC) pathotype. While strains belonging to virulence-associated lineages lacked key plasmid-encoded virulence plasmids, several plasmid replicons were detected in most of the E. coli (14/19) strains. Food contaminated with these pathogens can serve as a vehicle for disease transmission, posing a significant public health risk and necessitating stringent food safety and hygiene practices to prevent outbreaks. Hence, there is need for continuous surveillance and preventive measures to stop the spread of foodborne pathogens and reduce the risk of associated illnesses in Ghana.

Solanum pan-genomics and pan-genetics reveal paralogs as contingencies in crop engineering

Benoit, M. — 2024-09-14

Pan-genomics and genome editing technologies are revolutionizing the breeding of globally cultivated crops. A transformative opportunity lies in the reciprocal exchange of genotype-to-phenotype knowledge of agricultural traits between these major crops and hundreds of locally cultivated indigenous crops, thereby enhancing the diversity and resilience of our food system. However, species-specific genetic variants and their interactions with desired natural or engineered mutations pose barriers to achieving predictable phenotypic effects, even between closely related crops or genotypes. Here, by establishing a pan-genome of the crop-rich genus Solanum and integrating functional genomics and genetics, we show that gene duplication and subsequent paralog diversification are a major obstacle to genotype-phenotype predictability. Despite broad conservation of gene macrosynteny among chromosome-scale references for 22 species, including 13 indigenous crops, hundreds of global and lineage-specific gene duplications exhibited dynamic evolutionary trajectories in paralog sequence, expression, and function, including among members of key domestication gene families. Extending our pan-genome with 10 cultivars of African eggplant and leveraging quantitative genetics and genome editing, we uncovered an intricate history of paralog emergence and evolution within this indigenous crop. The loss of an ancient redundant paralog of the classical regulator of stem cell proliferation and fruit organ number, CLAVATA3 (CLV3), was compensated by a lineage-specific tandem duplication. Subsequent pseudogenization of the derived copy followed by a cultivar-specific structural variant resulted in a single fused functional copy of CLV3 that modifies locule number alongside a newly identified gene controlling the same trait. Our findings demonstrate that paralog diversifications over short evolutionary periods are critical yet underexplored contingencies in trait evolvability and independent crop domestication histories. Unraveling these contingencies is crucial for translating genotype-to-phenotype relationships across related species.

Dissecting earthworm diversity in tropical rainforests

Goulpeau, A. — 2024-09-19

Tropical rainforests are among the most emblematic ecosystems in terms of biodiversity. However, our understanding of the structure of tropical biodiversity is still incomplete, particularly for certain groups of soil organisms such as earthworms, whose importance for ecosystem functioning is widely recognised. This study aims at determining the relative contribution of alpha and beta components to earthworm regional diversity at a hierarchy of nested spatial scales in natural ecosystems of French Guiana. For this, we performed a hierarchical diversity partitioning of a large dataset on earthworm communities, in which DNA barcode-based operational taxonomic units (OTUs) were used as species surrogates. Observed regional diversity comprised 256 OTUs. We found that alpha diversity was lower than predicted by chance, regardless of the scale considered. Community-scale alpha diversity was on average 7 OTUs. Beta diversity among remote landscapes was higher than expected by chance, explaining as much as 87% of regional diversity. This points to regional mechanisms as the main driver of species diversity distribution in this group of organisms with low dispersal capacity. At more local scales, multiplicative beta diversity was higher than expected by chance between habitats, while it was lower than expected by chance between communities in the same habitat. This highlights the local effect of environmental filters on the species composition of communities. The calculation of a Chao 2 index predicts that as much as 1,700 species could be present in French Guiana, which represents a spectacular increase compared with available checklists, and calls into question the commonly accepted estimates of global number of earthworm species.

HDgraphiX: A web-based tool for visualization of hydrogen deuterium exchange mass spectrometry data

Vosper, K. R. — 2024-09-19

SummaryHydrogen deuterium exchange mass spectrometry (HDX-MS) investigates protein structural changes by measuring deuterium incorporation into the protein amide backbone. Due to richness of information provided on protein conformational dynamics, HDX-MS data can be challenging to visualize effectively. To address this, we have developed HDgraphiX, a web-based tool that visualizes HDX data by processing outputs from two popular analysis software packages, DynamX (Waters Corp.) and HDExaminer (Sierra Analytics Inc). HDgraphiX performs statistical analyses, filters data based on statistical significance and presents the results in several forms of user-friendly publication-quality heatmaps (Chiclet plots). Unique features of this tool include the generation of Woods plots, volcano plots, and PyMOL colouring scripts, which are used to map deuterium uptake differences onto protein structures. Additionally, HDgraphiX offers numerous advanced options for customizing data processing and plotting without the need for manual data editing. Availability and ImplementationHDgraphiX is available free of charge for all users at https://hdgraphix.net, the Python script and HTML template are deposited at https://github.com/KentV-UofG/HDgraphiX.

The mechanism of peptidoglycan O-acetylation in Gram-negative bacteria typifies bacterial MBOAT-SGNH acyltransferases

Anderson, A. C. — 2024-09-19

Bacterial cell envelope polymers are commonly modified with acyl groups that provide fitness advantages. Many polymer acylation pathways involve pairs of membrane-bound O-acyltransferase (MBOAT) and SGNH family proteins. As an example, the MBOAT protein PatA and the SGNH protein PatB are required in Gram-negative bacteria for peptidoglycan O-acetylation. The mechanism for how MBOAT-SGNH transferases move acyl groups from acyl-CoA donors made in the cytoplasm to extracellular polymers is unclear. Using the peptidoglycan O-acetyltransferase proteins PatAB, we explore the mechanism of MBOAT-SGNH pairs. We find that the MBOAT protein PatA catalyzes auto-acetylation of an invariant Tyr residue in its conserved C-terminal hexapeptide motif. We also show that PatB can use a synthetic hexapeptide containing an acetylated tyrosine to donate an acetyl group to a peptidoglycan mimetic. Finally, we report the structure of PatB, finding that it has structural features that shape its activity as an O-acetyltransferase and distinguish it from other SGNH esterases and hydrolases. Taken together, our results support a model for peptidoglycan acylation in which a tyrosine-containing peptide at the MBOATs C-terminus shuttles an acyl group from the MBOAT active site to the SGNH active site, where it is transferred to peptidoglycan. This model likely applies to other systems containing MBOAT-SGNH pairs, such as those that O-acetylate alginate, cellulose, and secondary cell wall polysaccharides. The use of an acyl-tyrosine intermediate for MBOAT-SGNH acyl transfer is also shared with AT3-SGNH proteins, a second major group of acyltransferases that modify cell envelope polymers.

Floral Reversion based micropropagation of Day-Neutral Cannabis sativa L.

Tabatabaei, D. S. — 2024-09-20

Micropropagation systems have been developed for photoperiod sensitive cultivars of cannabis, but there are currently no published methods for day-neutral genotypes. Most established cannabis micropropagation systems rely on shoot proliferation using modal explants that need them to be maintained in vegetative growth, which is not possible for day-neutral genotypes. Floral reversion, the process by which plants revert to vegetative growth from the generative stage, has been demonstrated as an alternative and potentially more efficient approach to the micropropagation of photoperiodic cannabis. The current study investigated if this process could be adapted to facilitate the micropropagation of day-neutral genotypes and overcome existing barriers. During the process, various factors such as photoperiod and light intensity may influence the efficiency of floral reversion. To evaluate this approach in day-neutral cultivars, various photoperiods were compared to assess the impact on reversion rate and subsequent growth. Excised florets obtained from in vitro day-neutral C. sativa cv. "Blue Auto Mazar x auto Blueberry" seedlings were subjected to five photoperiods including 12.0, 16.0, 18.0, 20.0, and 24.0 hours of light per day for four weeks. Reversion rates and time, shoot length, shoot number, and node number were measured. Statistical analyses revealed significant differences (p-values < 0.05) in terms of reversion time among photoperiod treatments. The highest reversion rate happened under the 16.0 hr photoperiod with 72% success. The highest shoot lengths were observed under 20.0 hr of photoperiods with a mean of 10.1 mm and the lowest shoot length belonged to 12.0 hr of photoperiods with a mean of 5.6 mm, respectively. Reverted shoots developed vegetatively for some time before initiating new flowers. However, during this period the shoots were successfully rooted in vitro and then acclimated to the growth chamber where they completed their life cycle, including seed production. This process was also successfully achieved from a 2-year old culture of C. sativa cv. "Blue Auto Mazar", demonstrating that it is feasible to use this approach for long term culture maintenance and multiplication. This study demonstrates that floral reversion can successfully be used to clonally propagate day-neutral cannabis plants and serves as a basis for developing large-scale clonal propagation and breeding strategies.

Mean daily temperatures can predict the thermal limits of malaria transmission better than rate summation

Shocket, M. S. — 2024-09-23

Temperature shapes the distribution, seasonality, and magnitude of mosquito-borne disease outbreaks. Mechanistic models predicting transmission often use mosquito and pathogen thermal responses from constant temperature experiments. However, mosquitoes live in fluctuating environments. Rate summation (nonlinear averaging) is a common approach to infer performance in fluctuating environments, but its accuracy is rarely validated. We measured three mosquito traits that impact transmission (bite rate, survival, fecundity) in a malaria mosquito (Anopheles stephensi) across temperature gradients with three diurnal temperature ranges (0, 9 and 12{degrees}C). We compared thermal suitability models with temperature-trait relationships observed under constant temperatures, fluctuating temperatures, and those predicted by rate summation. We mapped results across An. stephenesis native Asian and invasive African ranges. We found: 1) daily temperature fluctuation significantly altered trait thermal responses; 2) rate summation partially captured decreases in performance near thermal optima, but also incorrectly predicted increases near thermal limits; and 3) while thermal suitability characterized across constant temperatures did not perfectly capture suitability in fluctuating environments, it was more accurate for estimating and mapping thermal limits than predictions from rate summation. Our study provides insight into methods for predicting mosquito-borne disease risk and emphasizes the need to improve understanding of organismal performance under fluctuating conditions.

A Comparison of the Sexual Expression, Biomass, Cannabinoid Content, and Seed Production in XXX and XXY Triploid Cannabis

Paul, N. — 2024-09-25

Cannabis is a multi-billion dollar industry reliant on unpollinated genetically female (XX) plants, as pollination reduces cannabinoid yields and flower quality. The mechanism behind the sexual expression of C. sativa is unclear. This study examines the sexual characteristics of homo- and hetero-gametic triploid cannabis plants (XX(X/Y)) to understand the role of the Y chromosome in cannabis sex determination. Heterogametic triploids were produced by crossing a male diploid C. sativa cv. Durban Poison (2n = 2x = 18 + XY) with a tetraploid female C. sativa cv. Higher Education (2n = 4x = 36 + XXXX) confirmed by flow cytometry and PCR. Measurements include fresh and dry plant weight, dry flower weight, seed counts, seed weights, and cannabinoids quantified using liquid chromatography. Results showed 44.4% of plants were XXY (n = 8), with 75% presenting a monoecious phenotype (n = 6). Of these, 50% produced predominately male flowers (n = 3), while the rest produced predominately female flowers. Plants with predominately female flowers had higher yields and cannabinoids compared to predominantly male plants. XXX genotypes outperformed XXY in several metrics. This study enhances our understanding of C. sativa sex determination and offers insight into breeding and cultivation strategies utilizing triploid cannabis.

Exploring the use of environment-agent-based models for risk assessment of Great Lakes piping plovers

Edwards, B. P. — 2024-09-27

The Piping Plover Charadrius melodus is an endangered species of shorebird endemic to North America. This species has been the centre of many modelling studies in the last decade. One model type that has been underused in Piping Plover studies is agent-based modelling, which can be used as an accurate risk assessment tool in simulating effects of anthropogenic activities on a given animal species. Recent innovations in ecological modelling have given rise to the environmental agent-based model (enviro-ABM), which efficiently stores information about spatially indexed environmental cells and treat those as agents. This restricts computation to only focus on environmental cells containing the species we are studying, allowing for a more efficient simulation. Using Python, a high-level programming language popular in scientific computing, we develop an enviro-ABM to provide simulations of Piping Plover hatchling growth during a given breeding season. We experiment with increasing levels of human presence and human exclosure size and observe their effects on the growth rate of the simulated Piping Plover hatchlings. Our simulations showed a clear decrease in Piping Plover growth rate as anthropogenic presence increased in the simulated environment. However, when we added a 100 m human exclosure around the nest, the effects of the anthropogenic presence were mitigated at each level. We conclude that an enviro-ABM can be used to assist with conservation and management decisions, with the caveat that the model be constantly updated and informed with results of field studies, especially those pertaining to foraging and energetics of Piping Plovers.

Development of multi-species qPCR assays for a stress transcriptional profiling (STP) Chip to assess the resilience of salmonids to changing environments

Islam, S. S. — 2024-09-27

Ecologically and socio-economically important salmonid fishes in Canada are threatened by diverse environmental stressors. However, predicting species responses to environmental change requires understanding the underlying molecular mechanisms governing environmental stress tolerance. Developing advanced molecular genetic tools will provide opportunities to predict how salmonid fishes will respond to environmental stressors and assess their adaptive potential and vulnerability into the future. Here, we developed a panel of Taqman quantitative PCR (qPCR) assays designed to measure mRNA transcript abundance at selected candidate loci for use across salmonids. We designed and applied those assays for use in a high-throughput nanofluidic OpenArray Stress Transcriptional Profiling Chip (STP-Chip) capable of 2688 simultaneous qPCR at multiple gene loci (112 targets for 12 samples in duplicate). Using the nanofluidic STP-Chip, we tested these 112 multi-species qPCR assays using gill, liver and muscle tissue from eight species of salmonids across four genera. Of the selected 112 assays, 69 assays showed amplification in gill, 64 in liver, and 67 in muscle across all eight salmonid species. The percentage of assays that showed amplification across three tissues varied between genera: In general, Salmo, Oncorhynchus, and Salvelinus species showed a higher success rate than Coregonus species. Stress, circadian rhythm, apoptosis, growth-metabolism, and detoxification-relevant assays showed high success rates for amplification across all salmonid species for all three tissues. In contrast, neural plasticity, appetite regulation, osmoregulation, immune function, endocrine disruption, and hypoxia-relevant assays showed low success. Not surprisingly, we observed tissue-specific variation among qPCR amplification patterns. There were significant differences in mRNA transcript abundance among species across the four genera, but we did not see variation between species from the same genus. These qPCR assays can be used to design custom STP-Chips that can be used for quantifying stress in salmonid fish, improving health through more accurate diagnostic tests for disease, and monitoring adaptation to accelerated climate change regionally and globally.

Spatiotemporal variation in size-dependent growth rates in small isolated populations of Arctic charr (Salvelinus alpinus)

Mittell, E. — 2024-09-27

As a key life-history trait, growth rates are often used to measure individual performance and to inform parameters in demographic models. Furthermore, intra-specific trait variation generates diversity in nature. Therefore, partitioning out and understanding drivers of spatiotemporal variation in growth rate is of fundamental interest in ecology and evolution. However, this has rarely been attempted due to the amount of individual-level data required through both time and space, and issues with missing data in important covariates. Here we implemented a Bayesian state-space model using individual-level data from 20 populations of Arctic charr (Salvelinus alpinus) across 15 capture occasions, which allowed us to: (1) integrate over the uncertainty of missing recapture records; (2) robustly estimate size-dependence; and (3) include a covariate (water temperature) that contained missing data. Interestingly, although there was substantial spatial, temporal, and spatiotemporal variation in growth rate, this was only weakly associated with variation in water temperature and almost entirely independent of size, suggesting that spatiotemporal variation in other environmental conditions affected individuals across sizes similarly. This fine-scale spatiotemporal variation emphasises the importance of local conditions and highlights the potential for spatiotemporal variation in a size-dependent life-history trait, even when environmental conditions are apparently very similar.

What we can find in what's left behind: DNA metabarcoding of semi-aquatic insect exuviae

Dworatzek, S. — 2024-09-28

Semi-aquatic insects contribute to critical ecological functioning of freshwater habitats in both the aquatic and terrestrial phase of their life. Traditional methods of studying their emergence rely on the capture of insects as they emerge, and morphological identification with taxonomic keys. This is not only time consuming but can have large impacts on the study population, obstacles that can be removed by the use of DNA. This study investigated the potential of using exuviae collected from the water surface as DNA source. Both emergence trap samples and insect exuviae were collected from a constructed wetland and a small creek in southern Ontario. Metabarcoding provided a total of 40 samples with 254 distinct taxa from emergence traps (26 samples), and 135 from exuviae (14 samples). There were many similarities between both two sample types, especially for the rich chironomid diversity. Nonetheless, the exuviae samples were able to identify more orders containing semi-aquatic insects. Furthermore, they showed a higher level of diversity within these orders. This higher level of diversity seen in exuviae samples may be due to limitations of emergence traps, such as they only account for a small defined surface area. In contrast, exuviae are representing a much larger area and are free floating, thus collection of emerging taxa is not limited to the emerging site. We were able to show that identification of emerging aquatic insects through metabarcoding of exuviae is a useful method for the study of insect emergence.

FungAMR: A comprehensive portrait of antimicrobial resistance mutations in fungi

Bedard, C. — 2024-10-07

Antimicrobial resistance (AMR) is a global threat. To optimize the use of our antifungal arsenal, we need rapid detection and monitoring tools that rely on high-quality AMR mutation data. Here, we performed a thorough manual curation of published AMR mutations in fungal pathogens to produce the FungAMR reference dataset. A total of 501 papers were curated, leading to 35,792 mutation entries all classified with the degree of evidence that supports their role in resistance. FungAMR covers 95 species, 246 genes and 208 drugs. We combined variant effect predictors with FungAMR resistance mutations and showed that these tools could be used to help predict the potential impact of mutations on AMR. Additionally, a comparative analysis among species revealed a high level of convergence in the molecular basis of resistance, highlighting some potentially universal resistance mutations. The analysis also showed that a significant number of resistance mutations lead to cross-resistance within antifungals of a class, as well as between classes for certain mutated genes. The acquisition of fungal resistance in the clinic and the field is an urging concern. Finally, we provide a computational tool, ChroQueTas, that leverages FungAMR to screen fungal genomes for AMR mutations. These resources are anticipated to have great utility for researchers in the fight against antifungal resistance.

Cefepime-Taniborbactam and Ceftibuten-Ledaborbactam Maintain Activity Against KPC Variants that Lead to Ceftazidime-Avibactam Resistance

Myers, C. L. — 2024-10-11

Klebsiella pneumoniae carbapenemases (KPCs) are widespread {beta}-lactamases that are a major cause of clinical non-susceptibility of Gram-negative bacteria to carbapenems and other {beta}-lactam antibiotics. Ceftazidime combined with the {beta}-lactamase inhibitor avibactam (CAZ-AVI) has been effective for treating infections by KPC-producing bacteria, but emergent KPC variants confer resistance to the combination. Taniborbactam and ledaborbactam are bicyclic boronate {beta}-lactamase inhibitors under development with cefepime and ceftibuten, respectively, to treat carbapenem-resistant bacterial infections. Here, we assessed the effects of clinically important KPC-2 and KPC-3 variants (V240G, D179Y, D179Y T243M) on the antibacterial activity of cefepime-taniborbactam (FEP-TAN) and ceftibuten-ledaborbactam (CTB-LED) and examined catalytic activity and inhibition of these variants. FEP-TAN and CTB-LED were highly active against CAZ-AVI-resistant engineered E. coli strains expressing these variants. Purified KPC variants catalyzed more efficient CAZ hydrolysis than wild-type enzymes, and D179Y-containing KPC-3 variants additionally catalyzed more efficient FEP hydrolysis than wild-type KPC-3. All KPC variants poorly hydrolyzed CTB, and D179Y-containing variants demonstrated significantly higher affinity for CAZ than FEP or CTB. Second-order rate constants (k2/K) for inhibition of D179Y-containing KPC-2 variants were significantly reduced relative to wild-type KPC-2, with AVI most impacted. K2/K was less affected for D179Y-containing KPC-3 variants, and reflected robust inhibition by TAN, LED and AVI. Together, the findings illustrate a biochemical basis for greater FEP-TAN and CTB-LED antibacterial activity in KPC variant expression backgrounds relative to CAZ-AVI, whereby the boronate inhibitors have sufficient inhibitory activity, whilst FEP and CTB are poorer substrates and bind to the variant enzymes with reduced affinity compared to CAZ.

The ambiguity of "hybrid swarm": inconsistent definitions and applications in existing research

Campbell, J. N. — 2024-10-12

1Hybridization is common in wild taxa and often increases in frequency following anthropogenic disturbance to an environment. Next-generation sequencing techniques make genomic analysis of a large number of individuals feasible, vastly improving the analysis for and promoting a greater frequency of studies on hybridization. However, terminology surrounding hybridization can be inconsistent; in particular, the term "hybrid swarm" has been used extensively in the literature but lacks a consistent definition. In this paper, we conducted a comprehensive review of the literature that uses the term "hybrid swarm" in reference to hybridization between taxa and challenged putative definitions of the term. We found that the term "hybrid swarm" is used in a variety of contexts, including some contradictory to other literature, and that there is little consensus on what constitutes a hybrid swarm in terms of hybrid outcomes, frequency relative to disturbances, or duration of existence. We dissuade researchers from use of the term "hybrid swarm" and instead suggest more specific and clear terminology to describe aspects of hybridization. Consequently, we hope that this paper promotes consensus surrounding hybridization terminology and improves the quality of future research on hybridization.

Complex responses of soil prokaryotes, fungi and protists to prairie restoration on retired agricultural lands

Tosi, M. — 2024-10-12

Restoring native ecosystems on marginal croplands has many benefits but the impacts on belowground biodiversity are less clear, in part because the limiting factors regulating soil biota are complex and poorly described. Here, we studied how grassland prairie restoration of marginal croplands affected the diversity and composition of soil microbiota on 5 conventional farms from Ontario, Canada. Soil samples (0-15 cm) were collected from annually cultivated fields and adjacent planted perennial grassland where cultivation and chemical inputs had ceased several years previously. Following DNA extraction, we estimated bacterial and fungal abundance using quantitative PCR, and microbial diversity of prokaryotes, fungi and protists using amplicon high-throughput sequencing. Under both land uses, prokaryotic communities were dominated by Proteobacteria, Actinobacteria and Acidobacteria, fungal communities by Ascomycota, and protist communities by Rhizaria (TSAR), Evosea (Amoebozoa) and Chlorophyta (Archaeplastida). Prairie restoration did not have a consistent effect on soil microbial abundance, richness or evenness, which responses varied across farms. Microbial genetic and taxonomic community composition (i.e., sequence variant and genus level) were affected by land use, farm and the interaction between these two factors. Generally, prairie soils had higher relative abundance of Latescibacterota, Desulfobacterota, Acidobacteriota and Glomeromycota, and lower of Deinococcota, Chytridiomycota and Amoebozoa_X. In terms of differentially abundant fungal genera, prairies promoted more fungal plant symbionts, less saprotrophs and no plant pathogens. Interkingdom networks revealed changes in potential microbe-microbe associations with prairie restoration, with only 8 associations in common between land uses. The relationship between soil microbial diversity and physicochemical properties varied across microbial groups, diversity metrics and land uses. Our results evidence the complexity associated with restoring soils from agricultural land to natural ecosystems, with unspecified farm-specific factors (e.g., soil type, prairie species, management history) strongly modulating the response of different microbial groups and variables.

Biotic interactions and environmental filtering both determine earthworm alpha and beta diversity in tropical rainforests

Goulpeau, A. — 2024-10-15

O_LIUnderstanding the relative importance of biotic interactions, multiple environmental drivers, and neutral processes in shaping community diversity and composition is a central question for both theoretical and applied ecology. C_LIO_LIWe analysed a dataset describing 125 earthworm communities sampled in 10 localities in French Guiana. DNA barcodes were used to delimit operational taxonomic units (OTUs) that we considered as species surrogates to avoid the taxonomic deficit and calculate community-scale species richness and pair-wise Sorensen beta-diversity. We used log-ratio and generalised linear models to highlight the effects of biotic interactions and environment as drivers of alpha diversity, and generalised dissimilarity models to figure out the relative contribution of space and environment to beta-diversity at different spatial extents. C_LIO_LICommunity-scale alpha diversity was mainly explained by habitat filtering (soil texture) and interspecific competition that limit the number of locally co-existing species. C_LIO_LIBeta diversity between pairs of communities was mainly explained by distance when comparing communities in similar habitats, by topography and available soil phosphorus when comparing communities in different habitats, and by distance, elevation and climate when comparing all possible pairs of communities. C_LIO_LIWhile community composition is determined locally by neutral processes and environmental filtering, biogeographic processes linked to dispersal limitation and adaptation to local environment are the most influential on a regional scale. This highlights the complex interplay of dispersal limitation, biotic interactions and environmental filtering during the process of community assembly. C_LI

Cannabidiol (CBD) potentiates physiological and behavioral markers of hypothalamic-pituitary-adrenal (HPA) axis responsivity in female and male mice

Jenkins, B. W. — 2024-10-21

RationaleClinical literature indicates there may be a therapeutic use of cannabidiol (CBD) for stress-related disorders. Preclinical literature remains conflicted regarding the underlying neurobehavioral mechanisms, reporting mixed effects of CBD (increased, decreased, or no effect) on anxiety- and fear-related behaviors. Preclinical data demonstrated that CBD modulates hypothalamus-pituitary-adrenal (HPA) axis gene expression; it is unknown whether CBD changes HPA axis responsivity and how this relates to altered behavior. ObjectivesWe aimed to evaluate whether acute or chronic CBD administration would alter physiological and behavioral measures of HPA axis responsivity in male or female mice. MethodsC57BL/6 mice of both sexes were injected with vehicle or CBD (30 mg/kg, i.p.) daily for 26 days. Plasma corticosterone (CORT) levels were evaluated following dexamethasone suppression and adrenocorticotropin hormone stimulation tests after acute and chronic CBD exposure. After chronic CBD, mice were tested for anxiety-like behavior using an elevated plus maze (EPM) and associative fear learning and memory using a trace fear conditioning (FC) protocol. ResultsCompared to vehicle, CBD induced a state of HPA axis hyperactivation, an effect which was significant in males; it also normalized anxiety-like behavior in female mice classified as having HPA axis hypofunction and primed all female mice for enhanced conditioned responding. Significant sex differences were also detected: females had greater plasma CORT levels and HPA axis responsivity than males, exhibited less EPM anxiety-like behavior, and were more responsive during FC. ConclusionsCBD potentiated physiological and behavioral markers of HPA axis function and normalized anxiety-like behavior in a sex-specific manner. This observation has implications for cannabinoid-based drug development targeting individuals with stress-related disorders involving HPA axis hypofunction pathology.

Submaximal eccentric training during immobilization does not prevent serial sarcomere loss or impairments in mechanical function in old or young rats

Hinks, A. A. — 2024-10-21

The age-related loss of muscle mass is partly driven by a reduction in serial sarcomere number (SSN), and further SSN loss occurs during immobilization. SSN is associated with optimal force and power production and muscle passive tension, thus immobilization-induced SSN loss is especially a concern for older individuals who are often subjected to forced muscle disuse with illness and injury. We previously showed that submaximal eccentric resistance training increased SSN and improved muscle function in old rats. The present study investigated whether this training could prevent the losses of SSN and function when performed intermittently during immobilization. 10 old (32 months) and 10 young (8 months) rats underwent unilateral casting of the plantar flexors in a shortened position for 2 weeks. Thrice weekly, casts were removed for isokinetic eccentric resistance training. Pre- and post-training we assessed in-vivo maximum isometric torque at ankle angles corresponding to stretched and neutral muscle lengths, the passive torque-angle relationship, and isotonic power. The soleus and medial gastrocnemius were harvested for SSN measurements, with the untrained leg as a control. In old and young rats, muscles of the casted leg had lower muscle wet weights (-20-40%), physiological cross-sectional area (-16-20%), and SSN (-7-29%) than the control leg. Furthermore, maximum isometric torque (-37-46%) and isotonic power (-{approx}70%) decreased, and passive torque increased (+{approx}400%) from pre- to post-training for both age groups. Thus, irrespective of age, submaximal eccentric resistance training 3 days/week was ineffective for preventing the losses of muscle contractile tissue and mechanical function during casting.

The mitochondrial-targeted antioxidant SkQ1 prevents mitochondrial-linked apoptosis but not necroptosis or skeletal muscle atrophy in ovarian cancer

Khajehzadehshoushtar, S. — 2024-10-25

The degree to which mitochondrial-linked cell death pathways contribute to skeletal muscle atrophy during cancer remain unknown. Here, we combined a novel and robust mouse model of metastatic ovarian cancer with chronic administration of the mitochondrial-targeted antioxidant SkQ1 to determine the time-dependent and muscle-specific relationships of mitochondrial-linked apoptosis and necroptosis to the development of muscle atrophy in the type II B-rich gastrocnemius. Early-stage ovarian cancer reduced type II B fibre cross-sectional area in the gastrocnemius but did not alter mitochondrial H2O2 emission despite increased activities of mitochondrial-linked caspase-9 and-3 regulators of apoptosis. During late-stage ovarian cancer, sustained atrophy was associated with increased mitochondrial H2O2 emission potential in vitro, a greater probability of calcium-triggered mitochondrial permeability transition and increases in downstream caspase-9 and -3 activity. SkQ1 attenuated mitochondrial H2O2 emission and caspase-9 and -3 activity in late-stage ovarian cancer but did not prevent atrophy. Necroptosis markers were heterogeneous across time with total RIPK1 increasing during early-stage cancer which reverted to normal levels by late-stages while phosphorylated RIPK3 decreased below control levels. These discoveries indicate that preventing increases in mitochondrial-linked apoptotic caspase-9 and -3 activity during late-stage ovarian cancer with SkQ1 does not prevent atrophy of type II B fibres. Furthermore, necroptotic markers are inconclusive during cancer in this muscle type but are not modified by SkQ1. These results do not support a causal relationship between mitochondrial H2O2-linked apoptosis or necroptosis and atrophy in type II B fibres during ovarian cancer but do not rule out potential relationships in other muscle types. Key PointsO_LICancer increases mitochondrial reactive oxygen species (ROS) in skeletal muscle during atrophy, but the role of ROS in regulating cell death remains unknown. C_LIO_LIWe show that attenuating gastrocnemius mitochondrial ROS with the mitochondrial-targeted antioxidant SkQ1 prevented mitochondrial-linked pro-apoptotic caspase 9- and 3-activities but did not affect markers of necroptosis in a mouse model of ovarian cancer. C_LIO_LIReductions in gastrocnemius muscle fibre cross-sectional areas and the wet weights of several muscles were not prevented by SkQ1. C_LIO_LIThese findings demonstrate that mitochondrial ROS regulate apoptotic caspases but not necroptosis, and neither pathway is linked to gastrocnemius atrophy in mice with ovarian cancer. C_LIO_LIThe degree to which mitochondrial ROS-linked cell death pathways regulate muscle mass in other muscle types and cancer models requires further investigation. C_LI

Mitochondrial-targeted plastoquinone therapy ameliorates early onset muscle weakness that precedes ovarian cancer cachexia in mice

Delfinis, L. J. — 2024-10-25

Cancer cachexia, and the related loss of muscle and strength, worsens quality of life and lowers overall survival. Recently, a novel pre-atrophy muscle weakness was identified during early-stage cancer. While mitochondrial stress responses are associated with early-stage pre-atrophy weakness, a causal relationship has not been established. Using a robust mouse model of metastatic epithelial ovarian cancer (EOC)-induced cachexia, we found the well-established mitochondrial-targeted plastoquinone SkQ1 partially prevents pre-atrophy weakness in the diaphragm. Furthermore, SkQ1 improved force production during atrophy without preventing atrophy itself in the tibialis anterior and diaphragm. EOC reduced flexor digitorum brevis (FDB) force production and myoplasmic free calcium ([Ca2+]i) during contraction in single muscle fibers, both of which were prevented by SkQ1. Remarkably, changes in mitochondrial reactive oxygen species and pyruvate metabolism were heterogeneous across time and between muscle types which highlights a considerable complexity in the relationships between mitochondria and muscle remodeling throughout EOC. These discoveries identify that muscle weakness can occur independent of atrophy throughout EOC in a manner that is linked to improved calcium handling. The findings also demonstrate that mitochondrial-targeted therapies exert a robust effect in preserving muscle force during the early pre-atrophy period and in late-stage EOC once cachexia has become severe.

Adiponectin-receptor agonism prevents right ventricular tissue pathology in a mouse model of Duchenne muscular dystrophy

Gandhi, S. — 2024-10-25

Cardiac fibrosis during Duchenne muscular dystrophy (DMD) arises from cellular damage and inflammation and is associated with myofibre hypertrophy and metabolic dysfunction. However, the extent to which these relationships develop across all 4 cardiac chambers, particularly during early-stage disease, remains unknown. Here, we discovered that very young D2.mdx mice exhibit fibrosis exclusively in the right ventricle (RV) and left atrium. Concurrent cardiomyocyte hypertrophy and disorganization in the RV were related to a highly specific inflammatory signature of increased infiltrating pro-inflammatory macrophages (CD11b+CD45+CD64+F4/80+CCR2+), myofibre mitochondrial-linked apoptosis, and reduced carbohydrate and fat oxidation. This relationship did not occur in the left ventricle. Short-term daily administration of a peptidomimetic adiponectin receptor agonist, ALY688, completely prevented RV fibrosis, myofibre hypertrophy, infiltrating macrophages and mitochondrial stress as well as left atrial fibrosis. Our discoveries demonstrate early-stage cardiac tissue pathology occurs in a chamber-specific manner and is prevented by adiponectin receptor agonism, thereby opening a new direction for developing therapies that prevent tissue remodeling during DMD. TeaserHeterogeneous distribution of fibrosis, inflammation and metabolic dysfunction across the heart is prevented by adiponectin receptor agonism in a mouse model of Duchenne muscular dystrophy.

Territorial behavior as a route of social microbial transmission in an asocial mammal

Petrullo, L. — 2024-10-31

Microbial transmission is a major benefit of sociality, facilitated by affiliative behaviors such as grooming and communal nesting in group-living animals. The spread of microbial symbionts through these pathways, and their incorporation into host microbiomes, can enhance host health and fitness by contributing to pathogen protection and metabolic flexibility. Are pathways that facilitate microbial transfer across hosts also present in animals that do not form social groups because territoriality limits social interactions and prevents group formation? Here, we addressed this question by combining longitudinal sampling of individual gut microbial communities, demographic data, and dynamic behavioral and spatial measures of territoriality from a non-social, highly territorial small mammal: wild North American red squirrels (Tamiasciurus hudsonicus). As squirrel densities increased, individual gut microbial communities became richer and more phylogenetically diverse, while among-individual differences in composition decreased. This pattern was characterized primarily by increases in obligately anaerobic and non-sporulating taxa with little to no tolerance for oxygen-rich environments, suggesting social rather than environmental routes of transmission. Moreover, territorial intrusions--in which conspecifics were found on within an individuals territorial space--increased gut microbial diversity among individuals defending larger territorial spaces. Using an intrusion-based social network analysis, we found that that pairs with stronger social association (via intrusions) exhibited higher gut microbial similarity. Taken together, our findings provide some of the first evidence for social microbial transmission in a non-social species, and suggest that increased density and territorial behavior can diversify and homogenize host gut microbial communities despite social isolation.

Trends in Coupled Human-Environment Systems Modelling: A Scoping Review

Thampi, V. — 2024-11-12

Classical environmental models assume the influence of humans on environmental systems is constant. However, human and environmental systems respond to one another. As such, coupled human-environment systems (CHES) models have been developed and are becoming more widely studied. In this review, we analyze CHES modelling techniques and study systems over a decade (May 2009-April 2019). We utilized the PRISMA method to filter publications from both Web of Knowledge and PUBMED, yielding 92 relevant papers for our review. Publications more than doubled from the 5-year interval May 2009-December 2013 (28/92) to the 5-year interval January 2014-April 2019 (64/92). CHES models typically used either differential equations (DEs) (44/92) or agent-based models (ABMs) (28/92). We organized the included literature with respect to the technique used to represent human behaviour. We noticed a diversity of approaches in this respect, but primarily optimization techniques (28/92) and game theory (34/92). We noticed a substantial increase in publications using more highly structured models in the second 5-year interval. We attribute this to reduced technological barriers to developing more detailed models, and greater data availability. We discuss the realism of the models and their ability to capture real-world dynamics. Finally, we explore avenues for future research, and discuss unconventional routes such as online communities and artificial intelligence modelling to expand representation of human behaviour in CHES models.

Bridging the Python Training Gap for Bioscientists in Brazil: Improvements and Challenges

Crestana, G. S. — 2024-11-26

The rapid evolution of high-throughput technologies in biosciences has generated diverse and voluminous datasets, requiring bioscientists to develop data manipulation and analysis skills. Python, known for its versatility and powerful libraries, has become a crucial tool for managing these datasets. However, there is a significant lack of programming training for bioscientists in many countries. To address this knowledge gap among scientists in Brazil, the Brazilian Python Workshop for Biological Data was introduced several years ago, focusing on basic programming concepts and data handling techniques using popular Python libraries. Despite the progress and positive feedback from earlier editions, challenges persisted, necessitating continuous adaptation and improvement to meet the evolving needs of bioscientists.This work describes the advancements made in the 2021 and 2022 editions of the workshop and discusses new suggestions for its ongoing enhancement. Key innovations were introduced in the workshop structure and coordination, including the creation of new committees and the establishment of a code of conduct. Feedback forms were updated to enable real-time adjustments during the event, improving its overall effectiveness. The workshop also expanded its reach by increasing geographical diversity among participants. New didactic strategies, such as pair-teaching, code clubs, and the integration of information and communication technologies (ICTs), were implemented to enhance learning outcomes. Programming best practices and scientific reproducibility were emphasized through talks and hands-on activities, guided by PEP8 conventions. Furthermore, efforts to enhance scientific dissemination were intensified, with an increased presence on social media and participation in international scientific events and communication networks. Finally, we present updated recommendations for students, researchers, and educators interested in organizing and promoting similar events, building on those previously described.

Boosting of CAR-T cells with rhabdovirus is limited by type I interferon and rapid contraction

Burchett, R. — 2024-12-05

Rhabdovirus vaccines that encode tumour-associated antigens are potent boosting agents for adoptively transferred tumour-specific T cells. Employing rhabdovirus vaccines to boost adoptively transferred T cells relies on a priori knowledge of tumour epitopes, isolation of matched epitope-specific T cells, and a personalized vaccine, which limit clinical feasibility. Here, we investigated a universal strategy for boosting transferred tumour-specific T cells where boosting is provided through a chimeric antigen receptor (CAR) that is paired with a vesicular stomatitis virus (VSV) vaccine encoding the CAR-target. Boosting CAR-engineered tumour-specific T cells with paired VSV vaccines was associated with robust T cell expansion and delayed tumour progression in syngeneic models. CAR-T cell expansion and anti-tumour function was enhanced by blocking IFNAR1. However, vaccine-boosted CAR-T cells rapidly contracted and antigen-positive tumours re-emerged. In contrast, when the same T cells were boosted with VSV encoding antigen that stimulates through the TCR, the adoptively transferred T cells displayed improved persistence, tumour-specific endogenous cells expanded in parallel, and tumour cells carrying the antigen target were completely eradicated. Our findings underscore the need for further research into CAR-mediated boosting, how this differs mechanistically from TCR-mediated boosting, and the importance of engaging endogenous tumour-reactive T cells to achieve long-term tumour control.

HAPLODIPOIDY ACCELERATES MITOGENOME EVOLUTION IN INSECTS

Pakrashi, A. — 2024-12-05

Rates of mitogenome evolution differ among animal lineages, variation which has been linked to life history, ecological traits, and potentially to breeding system. Insects are a good model for examining the latter impacts as, although most are diplodiploids (DD), some lineages reproduce by haplodiploidy (HD) or thelytoky. In this study, we ask if breeding system influences patterns of evolution in the mitogenome using the 658 bp barcode region of the cytochrome c oxidase I (COI) gene as a sentinel. Specifically, we ask if the incidence of amino acid substitutions and indels is linked to breeding system. We investigated this matter by examining COI sequences from specimens assigned to 1332 BINs, a species proxy. Belonging to 513 families and 26 orders, these BINs included representatives of roughly half of all insect families. Most are DD, but ten lineages, varying in rank from tribe to order, are HD. Our analysis reveals that HD lineages show higher rates of amino acid substitution than DD. In addition, indels are frequent in HD lineages but are absent in DD taxa with one exception. Among DD, the Strepsiptera is an outlier; its COI shows very rapid amino acid evolution and frequent indels. The accelerated rates of mitogenome evolution in HD lineages suggest that this breeding system facilitates coevolution between the nuclear and mitochondrial genomes.

ROSLT: An Improved Raspberry-Pi Open-Source Live Voluntary-Wheel Running Tracker Method and Resource

Sanosa, P. — 2024-12-05

BackgroundExercise promotes health and has therapeutic effects on disease. Over time, the body improves its maximal exercise capacity through training adaptations such as an increase in VO2 max. In mice, voluntary wheel running allows for a natural setting to test spontaneous running behaviour under non-stressed conditions. There is a need to design sensitive animal-based assay that improves resolution for differentiating exercise performance from a regular cyclometer (which presents a single value from a summary of dynamic data collected over time) and offer circadian analyses. The purpose of this work is to examine the exercise behaviours of mice with a focus on circadian rhythm of running. We hypothesize the pi cyclometer (ROSLT) will mirror VDO M2.1 behaviors, enhancing circadian rhythm insights. MethodsUsing a hand-built cyclometer programmed through the Raspberry Pi computer, voluntary wheel running behaviours in CD-1 mice ([~]8-10 weeks) were recorded for 6 consecutive days. This features a Hall Effect sensor and neodymium magnets attached on the running wheels that will detect changes to wheel rotation, speed, acceleration, and distance (continuously) and publish the data to a server in real-time. To compare capabilities, running wheels will also be equipped with the VDO M2.1 WR Cycling Computer to track distance which will be manually recorded once a day. Accuracy from both devices were mechanically validated. ResultsThe main findings include that voluntary wheel running distance over 6 days produces inaccuracies by the VDO. The VDO showed fluctuations in distance over the last 3 days, while ROSLT showed consistent measurements. ConclusionThis comparison shows that ROSLT expands on the running activity of mice each day while maintaining accuracy and precision. This novel dynamic circadian cyclometer will advance our research abilities and can be used in differentiating exercise performance in applications such as doping.

Probing the structural heterogeneity of Pup ligase PafA using H/D exchange mass spectrometry

Plourde, A. — 2024-12-17

The Pup-proteasome system (PPS) is a unique bacterial proteolytic pathway found in some bacterial species, including in Mycobacterium tuberculosis, that plays a vital role in maintaining proteome integrity and survival during infection. Pupylation is the process of tagging substrates with Pup for degradation and is catalyzed by PafA, the sole Pup ligase in bacteria. However, how PafA interacts with diverse targets and its oligomeric state remain poorly understood. Although X-ray crystal structures have characterized PafA as a domain-swapped dimer, it is widely regarded as functionally active in its monomeric form. It remains to be established whether PafA dimerizes in solution, and how dimerization influences its function. In this study, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS) alongside complementary biophysical techniques to explore the oligomeric states and conformational dynamics of PafA. We show that recombinantly-produced PafA exists in a monomeric and a domain-swapped dimeric state in solution. While PafAdimer is stabilized by nucleotide, it predominately resides in a catalytically inactive conformation. Our HDX-MS highlighted regions throughout the N- and C-terminal domains that facilitate the PafA dimerization process. HDX-MS also revealed nucleotide binding induces global conformational changes on the PafAmonomer, underscoring the structural plasticity of this promiscuous enzyme. Our findings provide insight into the structure-function-dynamics relationship of PafA, shedding light on how its oligomeric states and conformational flexibility might influence its role in the PPS.

ProteoPlotter: an executable proteomics visualization tool compatible with Perseus

Olabisi-Adeniyi, E. — 2024-12-31

Mass spectrometry-based proteomics experiments produce complex datasets requiring robust statistical testing and effective visualization tools to ensure meaningful conclusions are drawn. The publicly-available proteomics data analysis platform, Perseus, is extensively used to perform such tasks, but opportunities to enhance visualization tools and promote accessibility of the data exist. In this study, we developed ProteoPlotter, a user-friendly, executable tool to complement Perseus for visualization of proteomics datasets. ProteoPlotter is built on the Shiny framework for R programming and enables illustration of multi-dimensional proteomics data. ProteoPlotter provides mapping of one-dimensional enrichment analyses, enhanced adaptability of volcano plots through incorporation of Gene Ontology terminology, visualization of 95% confidence intervals in principal component analysis plots using data ellipses, and customizable features. ProteoPlotter is designed for intuitive use by biological and computational researchers alike, providing descriptive instructions (i.e., Help Guide) for preparing and uploading Perseus output files. Herein, we demonstrate the application of ProteoPlotter towards microbial proteome remodeling under altered nutrient conditions and highlight the diversity of visualizations enabled with the platform for improved biological characterization. Through its comprehensive data visualization capabilities, linked to the power of Perseus data handling and statistical analyses, ProteoPlotter facilitates a deeper understanding of proteomics data to drive new biological discoveries.

Determination of the mechanisms of MCPA resistance in Amaranthus powellii

Aicklen, I. — 2025-01-07

Resistance to 2-methyl-4-chloro-phenoxyacetic acid (MCPA) was recently confirmed in a population of green pigweed (Amaranthus powellii) from Dresden, Ontario, Canada, with a resistance factor of 4.4. Resistance to synthetic auxin herbicides in Amaranthus species has previously been linked to non-target site resistance mechanisms with low-level resistance factors (<10). Based on this information an investigation into the mechanism of resistance to MCPA was conducted in this population of green pigweed. No significant differences in absorption, translocation, and metabolism of 14C-MCPA existed between the resistant and a susceptible population of green pigweed. An RNA-Sequencing experiment to identify differentially expressed genes also confirmed this result. Genes that were differentially expressed in the resistant population were linked to target site modifications. A single nucleotide polymorphism (SNP) conferring a leucine to phenylalanine substitution was identified in auxin response factor (ARF) 9. This mutation may be in the Phox and Bem1p (PB1) domain in ARF9 which facilitates the interaction between ARFs and Aux/IAA repressor proteins. The results demonstrate that the mechanism of resistance to MCPA is not a non-target site mechanism and may be linked to a target site modification. Specifically, a SNP in ARF9 could disrupt the interaction between ARF9 and other Aux/IAAs which could prevent ubiquitination of Aux/IAAs and subsequent lethal action of MCPA.

Learning a CoNCISE language for small-molecule binding

Erden, M. — 2025-01-13

Rapid advances in deep learning have improved in silico methods for drug-target interaction (DTI) prediction. However, current methods do not scale to the massive catalogs that list millions or billions of commercially-available small molecules. Here, we introduce CoNCISE, a method that accelerates drug-target interaction (DTI) prediction by 2-3 orders of magnitude while maintaining high accuracy. CoNCISE uses a novel vector-quantized codebook approach and a residual-learning based training of hierarchical codes. Strikingly, we find that much of binding-specificity information in the small molecule space can be compressed into just 15 bits of information per compound, characterizing all small molecules into 32,768 hierarchically-organized binding categories. Our DTI architecture, which combines these compact ligand representations with fixed-length protein embeddings in a cross-attention framework, achieves state-of-the-art prediction accuracy at unprecedented speed. We demonstrate CoNCISEs practical utility by indexing 6.4 billion ligands in the Enamine dataset, enabling researchers to query vast chemical libraries against a protein target in seconds. A "CoNCISE + docking" pipeline screened Enamine to propose strong binders (predicted KD {approx} 10-20 {micro}M) of three difficult-to-drug targets, each within two hours. CoNCISEs advance could democratize access to largescale computational drug discovery, potentially enabling rapid identification of promising molecules for therapeutic targets and cellular perturbations.

TaWUS2D regulates the number of grains per spikelet by enhancing the number of fertile ovaries in Multi-Ovary Wheat

Schoen, A. W. — 2025-02-08

Innovative genetic improvements in the staple crop Triticum aestivum (bread wheat) are urgently needed to address the growing global food security crisis. Here, we report the map-based cloning of TaWUS2D, the gene responsible for the dominant multi-ovary phenotype in wheat. Multi-ovary lines are characterized by the development of three fertile ovaries per floret that results in three grains, as opposed to wildtype single ovary wheat. We used HiFi long-reads to assemble a 14.48 Gbp genome scaffold assembly in the background of mutli-ovary wheat line MOV. Using high-resolution genetic mapping, combined with additional genomic resources, we defined the Mov-1 locus to a 135 Kbp region containing two genes. Using five independent deletion mutants and eight TILLING mutants, we demonstrate that a functional WUSCHEL-like protein, TaWUS2D, is required for the multi-ovary phenotype. TaWUS2D is upregulated in the MOV genetic background. This research lays the groundwork for developing new approaches to improve wheat production potential and sustainability in the face of current and future global food challenges.

WaveSeekerNet: Accurate Prediction of Influenza A Virus Subtypes and Host Source Using Attention-Based Deep Learning

Nguyen, H.-H. — 2025-02-28

BackgroundInfluenza A virus (IAV) poses a significant threat to animal health globally, with its ability to overcome species barriers and cause pandemics. Rapid and accurate IAV subtypes and host source prediction is crucial for effective surveillance and pandemic preparedness. Deep learning has emerged as a powerful tool for analyzing viral genomic sequences, offering new ways to uncover hidden patterns associated with viral characteristics and host adaptation. FindingsWe introduce WaveSeekerNet, a novel deep learning model for accurate and rapid prediction of IAV subtypes and host source. The model leverages attention-based mechanisms and efficient token mixing schemes, including the Fourier Transform and the Wavelet Transform, to capture intricate patterns within viral RNA and protein sequences. Extensive experiments on diverse datasets demonstrate WaveSeekerNets superior performance to existing models that use the traditional self-attention mechanism. Notably, WaveSeekerNet rivals VADR (Viral Annotation DefineR) in subtype prediction using the high-quality RNA sequences, achieving the maximum score of 1.0 on metrics including the Balanced Accuracy, F1-score (Macro Average), and Matthews Correlation Coefficient (MCC). Our approach to subtype and host source prediction also exceeds the pre-trained ESM-2 (Evolutionary Scale Modeling) models with respect to generalization performance and computational cost. Furthermore, WaveSeekerNet exhibits remarkable accuracy in distinguishing between human, avian, and other mammalian hosts. The ability of WaveSeekerNet to flag potential cross-species transmission events underscores its significant value for real-time surveillance and proactive pandemic preparedness efforts. ConclusionsWaveSeekerNets superior performance, efficiency, and ability to flag potential cross-species transmission events highlight its potential for real-time surveillance and pandemic preparedness. This model represents a significant advancement in applying deep learning for IAV classification and holds promise for future epidemiological, veterinary studies, and public health interventions.

Blue-shifted ancyromonad channelrhodopsins for multiplex optogenetics

Govorunova, E. G. — 2025-03-01

Light-gated ion channels from protists (channelrhodopsins or ChRs) are optogenetic tools widely used for controlling neurons and cardiomyocytes. Multiplex optogenetic applications require spectrally separated molecules, which are difficult to engineer without disrupting channel function. Scanning numerous sequence databases, we identified three naturally blue-shifted ChRs from ancyromonads. They form a separate branch on the phylogenetic tree and contain residue motifs characteristic of anion ChRs (ACRs). However, only two conduct chloride, whereas the closely related Nutomonas longa homolog generates inward cation currents in mammalian cells under physiological conditions, significantly exceeding those by previously known tools with similar spectral maxima (peak absorption at [~]440 nm). Measurements of transient absorption changes and pH titration of purified proteins combined with mutant analysis revealed the roles of the residues in the photoactive site. Ancyromonad ChRs could be activated by near-infrared two-photon illumination, a technique that enables the deeper-tissue optogenetic activation of specific neurons in three dimensions. Both ancyromonad ACRs allowed optogenetic silencing of mouse cortical neurons in brain slices. Ancyromonas sigmoides ACR (AnsACR) expression in cholinergic neurons enabled photoinhibition of pharyngeal muscle contraction in live worms. Overall, our results deepen the mechanistic understanding of light-gated channel function and expand the optogenetic toolkit with potent, blue-shifted ChRs. Impact statementAncyromonad channelrhodopsins advance our understanding of ionic selectivity and wavelength regulation in light-gated ion channels, and expand the toolkit for multiplexing with red-shifted fluorescent sensors.

Changes in serial sarcomere number of five hindlimb muscles across adult aging in rats

Hinks, A. — 2025-03-11

IntroductionAging is associated with a reduction in muscle fascicle length (FL), which contributes to sarcopenia: the age-related loss of muscle mass and function. Studies on rodents have confirmed this reduced FL is driven by a loss of sarcomeres aligned in series (serial sarcomere number; SSN) along a muscle. However, studies on rodents have focused primarily on rat plantar flexor SSN at two aging timepoints, leaving an incomplete view of age-related changes in SSN. Hence, this study investigated SSN as a contributor to the age-related loss of muscle mass in five hindlimb muscles across four aging timepoints in rats. MethodsThe soleus, medial gastrocnemius (MG), plantaris, tibialis anterior (TA), and vastus lateralis (VL) were obtained from 5 young (8 months), 5 middle-aged (20 months), 5 old (32 months), and 5 very old (36 months) male F344BN rats. After fixation of muscles in formalin and digestion in nitric acid, fascicles were teased out end-to-end to measure FL. SSN was determined by dividing FL by sarcomere length measured via laser diffraction. Muscle wet weight, anatomical cross-sectional area (ACSA), and physiological cross-sectional area (PCSA) were also determined for insight on age-related losses of whole-muscle mass and in-parallel muscle morphology. ResultsAge-related SSN loss was apparent after middle age for all muscles, with the plantaris showing the smallest (8%) and the VL the greatest (21%) loss. The MG and VL appeared to plateau in their SSN loss by 32 months, while the soleus and TA underwent continued loss from 32 to 36 months. In all muscles, SSN loss evidently contributed in part to the loss of muscle mass, alongside losses of contractile tissue in parallel (indicated by ACSA and PCSA). ConclusionAs SSN is closely tied to biomechanical function, these findings present SSN as a distinct target for improving muscle performance in older adults.

Arabidopsis root lipid droplets are hubs for membrane homeostasis under heat stress, and triterpenoid synthesis and storage.

Scholz, P. — 2025-03-26

O_LIPlant lipid droplets (LDs) and their associated proteins have numerous subcellular and physiological functions. While considerable progress has been made for LDs in many tissues, the function and composition of LDs in roots remains largely unexplored. C_LIO_LIWe investigated the changes of the number of LDs and of the lipidome in heat-stressed Arabidopsis thaliana roots. Furthermore, we isolated root LDs from the Arabidopsis mutant trigalactosyldiacylglycerol 1-1 sugar dependent 1-4 and investigated their proteome and lipidome. C_LIO_LIHeat stress lead to a degradation of membrane lipids and an increase in TAGs and LDs. while, fatty acid SEs decreased, probably acting as precursors for acylated sterol glycosides. A variety of proteins were enriched in root LDs, which are thus far not described as LD proteins. Transient expression of these proteins in many cases confirmed their LD localization, for example of the triterpene biosynthetic enzymes thalianol synthase and marneral synthase. We could furthermore show that the educts and products of these enzymes are enriched in root LDs, too. C_LIO_LIWe conclude that root LDs simultaneously act as a sink and source during heat stress-induced membrane remodeling. Furthermore, root LDs play a pivotal role in triperpene synthesis and storage, thereby highlighting LDs as hubs in specialized metabolism. C_LI

Deep learning using structural MRI massively improves prediction accuracy of body mass index

Cooper, A. — 2025-04-18

Obesity is a major public health problem globally and there is considerable interest in the neural mechanisms in food overconsumption. Artificial intelligence (AI), particularly machine learning, has shown promise in characterizing links between brain morphometry and obesity. In 1106 adults, compared to other forms of machine learning, deep learning using 3D convolutional neural networks (3D-CNN) dramatically improves prediction of body mass index (BMI). The 3D-CNN model robustly predicted BMI (R2 =.325), outperforming random forest, elastic net, and tabnet models (R2s<.07) in a lockbox sample. Explainable AI analyses revealed the specific brain regions implicated and these regions were moderately associated with delay discounting, fluid cognition, gait speed, dexterity, and alcohol use. Collectively, these findings reveal the value of deep learning for understanding of the neural basis and motivational processes in the neurobiology of obesity.

Factors Influencing Emerald Ash Borer Ecological Interactions

Borsato, N. D. — 2025-04-22

Emerald ash borer beetles (Agrilus planipennis) in North America are a destructive invasive species that increase tree mortality continent-wide, resulting in major ecological and economic impacts. Trees that are infested experience mortality rates which can exceed 99%, disrupting ecological communities and threatening the $218 billion forestry industry in North America. Given the ecological and economic impact of these pests, we seek to identify biological interactions and gain a better understanding of what ecological factors might influence these relationships. We use DNA metabarcoding from multiple markers to analyze the fungal, parasitic, plant, and microbial interactions of these beetles, and assess the relative importance of life stage (e.g., larvae, pupae, and adults), collection location, habitat, and date on the detection of ecological interactions. We detected 30 different taxonomic orders including 29 order-level interactions in larva1-stage individuals (3 animal, 17 bacteria, and 9 fungi), 64 in larva2-stage individuals (8 animal, 24 bacteria, and 32 fungi), 10 in larva3-stage individuals (2 animal, 3 bacteria, and 5 fungi), 74 in the pupae (5 animal, 31 bacteria, and 38 fungi), and 82 in the adult beetles (4 animal, 48 bacteria, 29 fungi, and 1 parasitic alveolate). These detections include several likely agents of biocontrol including the known commercially available Beauveria fungus, and several potential parasites including Wolbachia and ichneumonid wasps. A random forest model suggests the detection of interactions is best predicted by collection date and life stage, with interactions more likely to be detected in pupal samples which may be the ideal target for future analysis, where cost and time constraints prevent the more thorough analysis of all life stages.

Multiple variants of the mitochondrial COI DNA barcode region are prevalent in North European sawflies

Prous, M. — 2025-04-22

DNA barcoding, the use of standard segments of DNA to assign specimens to a species, has emerged as a major field of biodiversity research over the last 20 years. Large-scale global initiatives are building DNA barcode reference libraries for animals, fungi, and plants, while pipelines are being developed for metabarcoding-based biomonitoring. The effectiveness of these approaches rests on the premise that much less variation exists within species than between them. While exceptions occur, this principle has been demonstrated to apply in the many animal taxa where the barcode region of the COI gene is effective in species discrimination. Sawflies are an exception to this general pattern because DNA barcodes often fail to distinguish congeneric species, an observation which prompted us to search for an explanation. Using high-throughput single-molecule DNA sequencing to recover COI sequences from thousands of sawflies, we found that single individuals often possess multiple, seemingly functional, full-length DNA barcodes - a phenomenon not documented at similar prevalence in any animal taxon. While the evolutionary causes of multiple variants require further investigation, our observation is remarkable as it violates the one-barcode-one-specimen assumption. The presence of multiple variants of barcodes within individuals does not jeopardize the concept, but its occurrence does introduce a complexity for species inventories based on metabarcoding. They will overestimate the species count when barcode-based operational species units are used as species proxies. Similarly, reference libraries must consider how best to deal with the high frequency of multiple variants in sawflies and any other groups of organisms. Significance StatementDNA barcoding is revolutionizing biodiversity science by enabling the accurate identification of organisms, accelerating taxonomic workflows, and permitting DNA-based biomonitoring. The DNA barcode region for the animal kingdom, mitochondrial COI, is highly effective in discriminating species in almost all studied animal groups. However, the use DNA barcoding is sometimes complicated by the presence of nuclear pseudogenes (NUMTs) or by variants of the mitogenome itself (heteroplasmy) within individuals. By using high-throughput sequencing (HTS) to analyze thou-sands of specimens, we demonstrate that multiple, seemingly functional, full-length variants of the COI barcode region are frequent in North European sawflies. Since these variants are sometimes very divergent, it is important to consider the impact of this within-individual variability on studies based on DNA barcodes.

Automating the Curation of DNA Barcode Databases for Vascular Plants

Kolter, A. — 2025-04-29

Comprehensive, curated, and current DNA barcode reference databases are essential for both the identification of single specimens and for the interpretation of metabarcoding data. In the case of plants, nuclear (ITS) and plastid (rbcL, matK) markers are commonly utilized in union. Because the plastid regions are segments of protein-coding genes, their alignment and analysis are usually straightforward. By contrast, the assembly and validation of records for ITS is considerably more difficult for two reasons - the prevalence of indels and the presence of intraindividual variation. This complexity has provoked the development of several workflows to support the curation of reference databases for the internal transcribed spacer (ITS) region for plant barcoding. However, the pipelines used to create these databases lack functionalities which are essential to ensure a solid post-analytical validation. This paper presents a new workflow to address these shortcomings, with the goal of enhancing the reliability and accuracy of plant barcoding studies. We furthermore demonstrate that clustering of reference databases results in a substantial drop in the fraction of queries that gain a correct species-level assignment. By contrast, setting an acceptance threshold for identifications, based on the distance between query and match provides a meaningful reduction of error rates in incomplete reference databases.

A large global soil carbon sink informed by repeated soil samplings

Jia, R. — 2025-04-29

Partitioning the terrestrial carbon sink between vegetation and soil is crucial for predicting future climate change, but the role of soils remains poorly quantified. Here, we compiled 3,099 soil organic carbon time series spanning five decades. We found a global soil organic carbon sink of 1.83 {+/-} 0.9 (mean {+/-} SE) petagrams per year from 1992 to 2020, driven by extratropical young forests, boreal old forests, and grasslands, while trends in tropical ecosystems remain uncertain. Our findings suggest the net land sink resides almost exclusively belowground as soil carbon, emphasizing the global opportunity of soil conservation and restoration for climate mitigation.

Forecasting Extinction Risk using Thermal Performance Curves and Population Dynamic Modeling

Vasseur, D. A. — 2025-04-30

Thermal Performance Curves (TPCs) have become a popular tool for assessing the risks imposed by climate warming and variability on ectotherms. These assessments typically measure the match between an organism or populations TPC and the distribution of its current or future thermal environment as a proxy for extinction risk. However, extinction can occur even when the average thermal environment appears closely matched to a populations TPC because population dynamics can be very sensitive to thermal stress. Here, we develop a new metric for assessing extinction risk using a stochastic model of logistic growth as a foundation. We show that boundaries delimiting persistence and extinction regions of parameter space can be derived for the simple case where the intrinsic (Malthusian) growth rate r varies stochastically and that these boundaries continue to make reliable predictions when temperature T varies stochastically and the Malthusian growth rate is given by a thermal performance curve r (T). We accomplish this by combining theory with stochastic simulations of population dynamics and a laboratory experiment where populations of the single-celled protist Paramecium caudatum were cultured across different temporal means and variances of temperature. The measure of risk that we develop and validate is straightforward and easily applicable to any population for which the thermal performance of Malthusian fitness is known, allowing more rigorous identification of the risks imposed by warmer and more variable temperatures across the globe.

Cryo-EM structure of a type VI secretion system delivered membrane-depolarizing toxin involved in bacterial antagonism

Colautti, J. — 2025-04-30

Many Gram-negative bacteria use type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighboring competitor cells. Members of the VasX protein family, such as VasX from Vibrio cholerae and Tke5 from Pseudomonas putida, disrupt the inner membrane of target cells by forming ion-permeable channels that dissipate the proton motive force, thereby interfering with essential physiological processes. However, the molecular structure of any VasX family effector has remained unknown. Here, we present a cryo-EM structure of Ptx2, a recently identified VasX family effector exported by a T6SS of Pseudomonas aeruginosa. Our structure reveals that Ptx2 is an elongated, multi-domain protein that bears little resemblance to proteins of known function. Notably, the apparent flexibility of its domains suggests that Ptx2, like other membrane-depolarizing toxins, undergoes substantial conformational changes to facilitate membrane insertion. Guided by these predicted structural rearrangements, we used mutagenesis coupled with phenotypic assays to identify key features required for its toxic activity. Together, these findings provide the first molecular level insights into the structure and mechanism of VasX family effectors and expand our understanding of how these proteins contribute to interbacterial antagonism.

Synergistic Effect of Thermoneutral Housing and Chronotherapeutic PD-1 Blockade Overcomes Melanoma Resistance

Tang, J. — 2025-05-19

BackgroundImmune-checkpoint blockade transformed melanoma therapy, yet the B16-F10 model resists PD-1 inhibition. Sub-thermoneutral housing (22{degrees}C) causes cold stress, and dosing outside circadian immune peaks dampens anti-tumour immunity. We hypothesised eliminating both would render B16-F10 curable. MethodsC57BL/6 mice (n=40) with B16-F10 tumours were randomised to conventional (22{degrees}C; ZT22 PD-1 timing), chronotherapy only (22{degrees}C; ZT4), thermoneutrality only (30{degrees}C; ZT22), or "double-hit" (30{degrees}C; ZT4). Anti-PD-1 was given on days 3, 6, 9, 12. Tumour volume, weight, survival, and day-14 serum IFN-{gamma} were recorded. Synergy threshold {Delta} [≥] 20% was prespecified. ResultsDay 14 control tumours were 1207{+/-}48 mm3. Chronotherapy and thermoneutrality slowed growth (737{+/-}55 mm3 and 565{+/-}28 mm3 respectively; p<0.0001). The double-hit nearly halted progression (43{+/-}12 mm3, -96%; p<10{square}1{square}), yielding seven complete responses and 100% survival. IFN-{gamma} increased 8.8-fold versus control (430{+/-}24 vs 48{+/-}7 pg mL{square}1; p<10{square}1{square}). Bliss {Delta} was 25%, confirming synergy. ConclusionsRemoving cold stress and circadian mistiming unmasks curative PD-1 response in refractory melanoma. Available warming blankets and morning infusions suggest a practical tactic to rescue PD-1 efficacy in "cold" tumours. A factorial multicentre trial is warranted.

Testing for a wildlife reservoir of divergent SARS-CoV-2 in white-tailed deer

Crawshaw, L. — 2025-05-23

The 2021 discovery of a divergent lineage (B.1.641) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in white-tailed deer (Odocoileus virginianus) from Ontario raised concerns that deer were a potential reservoir. To assess whether white-tailed deer continued to be infected with B.1.641 and to test for spillover into other species, we established a surveillance program in Ontario by sampling wildlife via existing monitoring programs and through active surveillance of captive and wild animals. Between 2022 to 2024, we tested 2,839 animals, identifying one active SARS-CoV-2 infection (a likely spillover of a recombinant XBB.2.3.11.3 lineage), but no cases of B.1.641. Overall, 93 animals (6.8%) tested positive for SARS-CoV-2 antibodies, including 89 white-tailed deer, two Virginia opossums (Didelphis virginiana), one American mink (Neogale vison), and one river otter (Lontra canadensis). In Southwestern Ontario, where B.1.641 was originally detected, 15.2% of deer samples were seropositive. Generalized Linear Models demonstrated that seropositive deer were more likely to be found in areas with a higher fall deer harvest and human population density, and closer to previous B.1.641 cases. Our data suggest that deer-associated B.1.641 may have caused a relatively localized epizootic without forming a stable reservoir. This study underscores the importance of One Health-focused surveillance.

Physics-Informed Digital Twin Can Predict Cerebral Blood Flow and Cerebral Vascular Regulation Mechanisms in Neurocritical Care Patients

Briggs, J. K. — 2025-05-23

1Cerebral blood flow is vital for brain function and is acutely controlled through a set of physiological mechanisms known as cerebral vascular regulation (CVR)[1-3]. It remains challenging to directly measure the dynamics and function of individual CVR mechanisms[4], limiting our ability to understand and optimize brain perfusion, particularly for neurologically injured patients. Digital twins offer an ideal tool for overcoming this gap because they enable estimation, tracking, and forecasting of unmeasured physiological states[5, 6]. Here, we introduce CereBRLSIM (Cerebral Blood Regulation Latent State Inference and Modeling), a digital twin that integrates physiological knowledge and patient data to infer CVR function and predict cerebral dynamics. Using both in vivo experiments and simulated data, CereBRLSIM predicted cerebral hemodynamics with high accuracy and estimated the dynamics of myogenic, endothelial, and metabolic mechanisms underlying CVR. When personalized to neurocritical care patient data, CereBRLSIM differentiated cerebral hemodynamic phenotypes, predicted patient outcomes, and forecasted blood flow with significantly higher accuracy than machine learning models. This work provides a novel, interpretable, and clinically compatible approach for quantifying CVR function and forecasting cerebral blood flow, enabling new opportunities in precision diagnostics and foundational understanding of cerebral hemodynamics.

Whole blood proteome dynamics defines predictive diagnostic and prognostic signatures of cryptococcal infection

Geddes-McAlister, J. — 2025-05-29

Across the globe, fungi are impacting the lives of millions of people through the development of infections ranging from superficial to systemic with limited treatment options. To effectively combat fungal disease, rapid and reliable diagnostic methods are required, including current methodologies using antigen detection, culturing, microscopy, and molecular tools. However, the flexibility of these platforms to diagnose infection using non-invasive methods and predict the outcome of disease are limited. In this study, we apply state-of-the-art mass spectrometry-based proteomics to perform dual perspective (i.e., host and pathogen) profiling of cryptococcal infection. Whole blood collected over a temporal scale following murine model challenged with the human fungal pathogen, Cryptococcus neoformans, detected >3,000 host proteins and 160 fungal proteins. From the host perspective, temporal regulation of known immune-associated proteins, including eosinophil peroxidase and lipocalin-2, along with suppression of lipoproteins, demonstrated infection- and time-dependent host remodeling. Conversely, from the pathogen perspective, known and putative virulence-associated proteins were detected, including proteins associated with fungal extracellular vesicles and host immune modulation. We also observed and validated a new mechanism of immune system response to C. neoformans through modulation of haptoglobin. Further, we assessed the predictive power of dual perspective proteome profiling toward prognostics of cryptococcal infection and report a previously undisclosed integration among virulence factor production, immune system modulation, and individual model survival. Together, our findings pose novel biomarkers of cryptococcal infection from whole blood and highlight the potential of personal proteome profiles to determine the prognosis of cryptococcal infection, a new parameter in fungal disease management.

Spatiotemporal dynamics of cryptococcal infection reveal novel immune modulatory mechanisms and antifungal targets

Woods, M. — 2025-05-30

The threat and incidence of fungal diseases are increasing, as is the severity and mortality rates associated with these infections. New strategies to combat fungal infections are urgently needed to overcome rising rates of resistance and the emergence of new pathogens. To promote invasion within a host, fungi use highly adapted and regulated virulence factors, and, in turn, the host adopts an active and dynamic immune response to suppress infection. Understanding the interplay between these processes is crucial to move fungal disease management and treatment forward and improve global health outcomes. Within the present study, we tackle these challenges using state-of-the-art mass spectrometry instrumentation to explore proteome remodeling during active infection of Cryptococcus neoformans at an unprecedented depth with spatiotemporal resolution. Our prioritization of three host organs (i.e., lungs, brain, spleen) critical to initiation, progression, and response of disease discovers tissue-specific remodeling across time. Within the lungs, we revealed early and sustained activation of the host immune response integrated with characterization of a promising new antifungal target, and we propose the discovery of a competitive inhibitor for functional target disruption. Within the brain, proteome remodeling aligns with disease progression, and we define a new mechanistic role for haptoglobin in fungal cell modulation, as well as showcasing an adaptive survival response of C. neoformans within an hypoxic environment. Within the spleen, we reveal new dynamics of immune system activation upon cryptococcal infection. Overall, we provide the deepest integrated view of cryptococcal disease dynamics across temporal and spatial scales, revealing unrecognized mechanisms of host immunity and fungal pathogenesis that offer new avenues for targeted therapeutic intervention and disease management.

Antimicrobial Susceptibility and Genomic Determinants of Resistance and Virulence in Mycoplasma cynos and Mycoplasma felis

Framst, I. — 2025-06-03

Mycoplasma cynos and Mycoplasma felis are important respiratory pathogens in dogs and cats. Due to the challenges of culturing these fastidious bacteria, little is known about their antimicrobial susceptibility or mechanisms of pathogenicity. Treatment is typically empirical, as in vitro antimicrobial activity has not been evaluated, and therapeutic efficacy remains unclear. This study aimed to assess in vitro susceptibility and identify genetic markers of antimicrobial resistance (AMR) and virulence in M. cynos and M. felis clinical isolates. Minimum inhibitory concentrations (MICs) for doxycycline, tetracycline, minocycline, enrofloxacin, marbofloxacin, and azithromycin were determined using a broth microdilution assay developed for this study. Hybrid genomes were generated using Oxford Nanopore and Illumina sequencing. AMR-associated single-nucleotide polymorphisms (SNPs) in the gyrA gene correlated with high MICs to enrofloxacin and marbofloxacin in both species. Mutations in 23S rRNA were associated with reduced susceptibility to azithromycin. In M. felis, novel variants in gyrA and the 50S ribosomal protein L4 were linked to decreased susceptibility to fluoroquinolones and azithromycin, respectively. The data also suggest potential intrinsic resistance to azithromycin in M. felis. All isolates required low MICs of tetracyclines, and resistance mutations were not identified in the 16S rRNA gene, supporting tetracyclines as effective first-line treatment options. Virulence genes, particularly those associated with adhesion and immune evasion, were detected in both M. cynos and M. felis. This study presents the first comprehensive genomic and phenotypic analysis of AMR and virulence in M. cynos and M. felis, providing new insights into their pathogenicity and informing evidence-based therapeutic strategies.

Global proteomic analysis of Cryptococcus neoformans clinical strains reveals significant differences between latent and lethal infection

Betancourt, J. J. — 2025-06-03

To predict the outcomes of disseminated fungal disease, a deeper understanding of host-pathogen interactions at the site of infection is needed to identify targets for clinical intervention and diagnostic development. Cryptococcus neoformans is the causative agent of cryptococcosis, the largest infectious killer of individuals living with HIV. Cryptococcal infection begins in the lungs, with loss of immunological control leading to disseminated central nervous system disease and death. Using advanced mass spectrometry-based proteomic techniques, in vivo infection models, and patient-derived clinical strains, we explored the proteomic profiles of C. neoformans infections related to differences in strain virulence. Our findings reveal that non-lethal latent infection produces a proteomic response that differs significantly from the response caused by lethal infections, and that the proteomic profiles of typical and hypervirulent infections are markedly similar despite differences in time-to-death. Overall, the mouse pulmonary proteomic response in latent infection is defined by enrichment of proteins and pathways involved in extracellular matrix organization, cell adhesion, and structural changes, while the lethal infection is dominated by host-defense, translation, and metabolic processes. These results provide clinically relevant information on how infections caused by different Cryptococcus strains may produce significantly different outcomes. We also identified abundant fungal proteins that could be future drug targets in latent and lethal cryptococcal infection. IMPORTANCECryptococcus neoformans is a fungal pathogen that causes substantial morbidity and mortality in immunocompromised individuals. The initial infection begins in the lungs after exposure to inhaled spores after which local immune cells respond by either killing or containing the fungal cells. Immunosuppression weakens the immune system and allows fungal cells in the lungs to escape through the circulatory system and invade the central nervous system and cause fatal disease. However, differences between fungal strains influence the severity of disease manifestation. Our group has previously described genetic differences that contribute to strain-specific disease manifestations. In this study, we expanded our analysis to investigate the proteomic differences between strains of C. neoformans to identify candidate proteins and pathways that contribute to disease manifestation. We found that latent infection differs significantly from lethal disease from both the host and pathogen proteomic perspectives and have identified several fungal protein targets for future study.

Life at the extremes: Maximally divergent microbes with similar genomic signatures linked to extreme environments

Safari, M. — 2025-06-06

Extreme environments impose strong mutation and selection pressures that drive distinctive, yet understudied, genomic adaptations in extremophiles. In this study, we identify 15 bacterium-archaeon pairs that exhibit highly similar k-mer-based genomic signatures despite maximal taxonomic divergence, suggesting that shared environmental conditions can produce convergent, genome-wide patterns that transcend evolutionary distance. To uncover these patterns, we developed a computational pipeline to select a composite genome proxy assembled from non-contiguous subsequences of the genome. Using supervised machine learning on a curated dataset of 693 extremophile microbial genomes, we found that 6-mers and 100 kbp genome proxy lengths provide the best balance between classification accuracy and computational efficiency. Our results provide conclusive evidence of the pervasive nature of k-mer-based patterns across the genome, and uncover the presence of taxonomic and environmental components that persist across all regions of the genome. The 15 bacterium-archaeon pairs identified by our method as having similar genomic signatures were validated through multiple independent analyses, including 3-mer frequency profile comparisons, phenotypic trait similarity, and geographic co-occurrence data. These complementary validations confirmed that extreme environmental pressures can override traditionally recognized taxonomic components at the whole-genome level. Together, these findings reveal that adaptation to extreme conditions can carry robust, taxonomic domain-spanning imprints on microbial genomes, offering new insight into the relationship between environmental mutagenesis and selection and genome-wide evolutionary convergence.

The neuroendocrine peptide catestatin promotes clearance of cutaneous Staphylococcus aureus through mast cell Mrgpr activation

Guth, C. — 2025-06-17

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of cutaneous infections, underscoring the need for alternative therapeutic strategies. Catestatin, a neuroendocrine antimicrobial peptide produced by neurons and epithelial cells, has been implicated in skin defense against pathogens such as MRSA, though its mechanisms remain unclear. Here, we show that catestatin expression is upregulated in MRSA-infected skin wounds and that topical catestatin application significantly reduces MRSA burden in infected murine cutaneous wounds. This effect is dependent on the mast cell-specific G protein-coupled receptor Mrgprb2, the murine ortholog of human MRGPRX2. Notably, catestatin treatment leads to Mrgprb2-dependent suppression of inflammatory cytokine production and leukocyte infiltration, alongside upregulation of the antimicrobial peptide Defb14. In human mast cells, catestatin induces MRGPRX2-dependent degranulation, histamine release, prostaglandin D2 production, and cytokine expression. Pharmacological inhibition and western blot analysis reveal that catestatin activates multiple downstream G protein-dependent signaling pathways in an MRGPRX2-dependent manner. These findings demonstrate that catestatin promotes bacterial clearance by activating mast cells through Mrgprb2, thereby enhancing antimicrobial peptide production. Our study positions catestatin as a promising mast cell-targeting immunotherapeutic candidate for treating antibiotic-resistant skin infections.

Genome-wide Pervasiveness and Localized Variation of k-mer-based Genomic Signatures in Eukaryotes

Sadjadi, N. — 2025-06-17

Genomic signatures are taxon-specific patterns in nucleotide sequence composition observed across different regions of a genome, used in the taxonomic classification of organisms and in inferring their evolutionary relationships. However, the nature and extent of the pervasiveness of a genomic signature across the expanse of a Telomere-to-Telomere (T2T) assembly, especially across the functionally diverse sequence elements and highly repetitive regions, remain counterintuitive and underexplored. This study aims to bridge this knowledge gap by systematically investigating the pervasiveness and variation of the genomic signature across the human genome and the genome of each of three other eukaryotic species from different kingdoms. Using the alignment-free k-mer-based Frequency Chaos Game Representation (FCGR) of DNA sequences, this study qualitatively and quantitatively analyzes the variations of the genomic signature along an entire genome. Qualitative analysis is first performed through visual inspection of FCGR patterns across different chromosomes of a species. In parallel, a quantitative analysis evaluates the variation of the genomic signature within a genome by comparing eight distance measures to identify the optimal one for the datasets in this study. By taking an intragenomic perspective with detailed analysis of chromosome landscapes these analyses reveal that, while the genomic signature is preserved in most genomic regions, exceptions exist in localized regions, such as tandem repetitions of short and long repeat units. Upon determining this pervasiveness, we assemble novel pipelines aimed at selecting a short contiguous representative genomic segment that encapsulates the sequence composition patterns characteristic of the entire genome. These representative segments are then used to assess intragenomic variation of the genomic signature, demonstrating that only a small proportion of segments (namely those characterized by regional density of short and long tandem repeats) show high distance values from the representative. No-tably, in the human genome, 80% of the segments have a distance of less than 0.24 (on a [0,1] DSSIM scale) from the representative. Moreover, we demonstrate that using these representative segments improves down-stream tasks, e.g., increasing one-nearest-neighbor (1-NN) taxonomic classification accuracy by 7% compared to selecting a random genomic segment to serve as a proxy of the genome. Lastly, this study presents a special-purpose graphical user interface (GUI) software tool, CGR-Diff, designed to provide both visual and quantitative comparisons of FCGRs of sample or user-provided DNA sequences, thereby facilitating intragenomic variation analysis of genomic signature within and across species.

Gene expression profile dynamics of earthworms exposed to ZnO and ZnO:Mn nanomaterials

van Lingen, H. J. — 2025-06-20

Zinc oxide containing nanomaterials may elicit toxic responses in environmental organisms such as earthworms. Although toxic responses in earthworms have been reported, few studies have attempted to understand the molecular mode of action dynamics explaining the observed toxicity at a transcriptional level. This study investigates the time-dependent gene expression response in earthworms exposed to ZnO nanomaterials and ZnO:Mn multicomponent nanomaterials. Earthworms were exposed to ZnO or ZnO:Mn for 7 days and to ZnO:Mn or MnCl2 for 14 days. Strong differential gene expression responses were observed after 4 days of exposure to ZnO nanomaterials and after 2 days of exposure to ZnO:Mn. Moderate differential gene expression responses were observed after 14 days of exposure to ZnO:Mn and MnCl2. Gene ontology (GO) enrichment analysis revealed that differentially expressed genes in earthworms exposed to ZnO were associated with terms such as actin, (striated) muscle cells, contractile fiber, myofibril, sarcomere and supramolecular cellular components. In addition, genes that were upregulated after 2 days of exposure to ZnO:Mn were linked to GO terms including cilium, microtubule, cell projection, axoneme and sperm flagellum cellular components. Downregulated genes were enriched to GO terms related to ribosomes, mitochondria, translation, peptide processes, respiration and oxidative phosphorylation. Finally, for earthworms exposed to ZnO:Mn and MnCl2 for 14 days, only a limited number of differentially expressed genes were involved in GO terms related to diverse biological implications. In summary, exposing earthworms to the ZnO and ZnO:Mn nanomaterials elicited a transient response in differential gene expression related to muscle biology and energy metabolism and translation that had largely disappeared by day 14 from the start of the exposure.

Mycotoxin-driven proteome remodeling reveals limited activation of Triticum aestivum responses to emerging chemotypes integrated with fungal modulation of ergosterols

Ramezanpour, S. — 2025-07-01

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is a globally important wheat disease reducing yield and grain quality. The pathogen produces mycotoxins deoxynivalenol (DON), 3-acetyl DON (3ADON), 15-acetyl DON (15ADON), and nivalenol (NIV), which threaten food and feed safety. During the past 15 years, surveillance has identified nove trichothecenes 3ANX and NX, which show increased virulence compared to DON. In this study, we investigated the effects of 15ADON/3ANX chemotype on both wheat and F. graminearum proteomes to identify proteins and pathways responsive to the emerging mycotoxin chemotype. We defined a core wheat proteome across all strains (15ADON- and 15ADON/3ANX-producing, and untreated controls) to explore changes in protein abundance associated with defense response, grain development, and reduced photosynthesis upon infection. Conversely, we identified 32 wheat proteins exclusively produced in the presence of 15ADON/3ANX strains, providing further insight into chemotype-specific responses of wheat. Additionally, assessment from the fungal perspective, reported 119 proteins exclusive to the 15ADON/3ANX strains, including those associated with virulence and mycotoxin production. Lastly, investigation of strain-specific proteome changes showed a significant reduction in mycotoxin protective mechanisms in wheat upon exposure to two 15ADON/3ANX strains, as well as a novel connection between elevated ergosterol biosynthesis and 15ADON/3ANX producing strains. Together, our study characterizes distinct protein production profiles in wheat and F. graminearum in response to 3ANX and provides evidence that these molecular changes influence fungal virulence and host defense responses.

Prenatal cannabis smoke exposure alters placental development in a murine model of pregnancy

Podinic, T. — 2025-07-05

Cannabis use during pregnancy continues to increase with smoking remaining the most common mode of consumption. While clinical studies highlight an association between prenatal cannabis use and adverse pregnancy outcomes, less is known about placental outcomes, even though many of the reported pregnancy outcomes are thought to be mediated via placental dysfunction. Here, we established a mouse model of gestational cannabis smoke exposure to investigate the impacts on fetal outcomes and placental structure and function. Pregnant CD1 mice were exposed daily to {Delta}9-tetrahydrocannabinol (THC)-dominant cannabis smoke (12-14% THC, 0-2% CBD) or filtered air from embryonic day (E)6.5 to E18.5 or parturition. Cannabinoid analyses in cannabis smoke-exposed, paired maternal and fetal livers revealed total THC and 11-Nor-9-carboxy-THC (THCA) concentrations of 135.95 {+/-} 13.60 ng/g and 30.84 {+/-} 4.68 ng/g, respectively. Moreover, Cyp1a1, a smoke-inducible enzyme, was induced by 4-fold in cannabis smoke-exposed placentae. No changes in offspring body weights were observed; however, there was a marked decrease in the brain-to-body weight ratio of exposed postnatal day 1 (PND1) offspring. Placentae from exposed dams were significantly reduced in size, with altered zonation marked by a significantly decreased junctional zone and increased labyrinth zone. Key trophoblast differentiation markers (Tfap2c, Tpbpa, Pcdh12) and placental endocrine regulators (Pl2, Igf1r) were significantly downregulated following cannabis smoke exposure in placentas. Furthermore, transcript levels of placental nutrient and vascularization markers, Glut1, Vegfa and Pparg were significantly decreased in cannabis smoke-exposed placentas. By employing a physiologically relevant platform of prenatal cannabis exposure in vivo we demonstrate the adverse effects of prenatal cannabis smoke exposure on placental structure and function as well as on fetal brain growth.

Preclinical Study of Cannflavins A and B Action Against Glioblastoma Cells

Holborn, J. — 2025-07-05

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackgroundC_ST_ABSFlavonoids represent a large group of naturally occurring polyphenolic compounds, many of which have been found to produce valuable biological outcomes, including action against cancer cells. Glioblastoma multiforme (GBM) is an aggressive type of brain tumor that is associated with a poor prognosis and has limited treatment options. Previous findings suggest that cannflavins can produce promising effects for pancreatic and bladder cancers, but the efficacy of these phytochemicals in attacking brain tumour cells remains unknown. Our study evaluates the potential of cannflavin A and cannflavin B against GBM cells using a range of in vitro approaches. ResultsWe conducted experiments using A-172 and U-87 GBM lines to assess the impact of cannflavins A and B on cell viability, cycle, migration, and invasion capacity. Our results revealed a consistent dose-dependent decrease in cell viability in both lines after the addition of cannflavin B to the culture media. Interestingly, we found that chrysoeriol (the non-prenylated synthesis precursor of cannflavins in the Cannabis sativa plant) only has a limited adverse effect on survival using the same approach, while techtochrysin (an O-methylated flavone) has none. Using time-lapse live-cell imaging and a scratch assay, we also show that cannflavins can inhibit tumor cell migration at concentrations below those that produce significant cell death. Finally, we found that cannflavin B exhibits anti-migratory and anti-invasive properties in transwell and tumorsphere assays, underscoring its multifaceted therapeutic potential. ConclusionThese findings suggest that cannflavin B holds promise as a therapeutic agent in the treatment of GBM. HO_SCPLOWIGHLIGHTSC_SCPLOWO_LICannflavin B but not cannflavin A limits the viability of A-172 and U-87 GBM cells in a concentration- and time-dependent manner. C_LIO_LIUsing time-lapse live-cell imaging and a scratch assay, we provide evidence that cannflavins A and B can inhibit GBM cell migration at concentrations below those that result in a significant reduction in cell viability. C_LIO_LICannflavin B exhibits robust anti-migratory and anti-invasive properties in transwell and tumorsphere assays, underscoring its multifaceted therapeutic potential. C_LIO_LIAlthough previous work had reported the capacity of cannflavins A and B to interfere with TrkB and downstream the MAPK/AKT signaling pathways in mouse primary neurons, the effect of those molecules is limited in GBMs, suggesting that other targets are engaged to produce the cellular effects. C_LI

A new phylogeny and phylogenetic classification for Solanaceae

Deanna, R. — 2025-07-12

Solanaceae are a globally distributed family of considerable economic importance. Recent phylogenetic work on various clades in the family has resulted in a plethora of names at various taxonomic levels. This results in a confusing set of names applied to clades at different ranks, and to the use of informal names that are often not accepted in online resources. To solve this problem, we present here a phylogenetic classification following the PhyloCode for the Solanaceae. Traditional taxon names previously recognized at the ranks of tribe, genus, and section were maintained when associated with clades. We base our clade definitions on a newly constructed phylogeny of the family using 10 plastid and nuclear markers with a broad sampling of 1,474 species, roughly half of the diversity of the family. We complement this marker-based analysis with a phylogenomic analysis of 110 taxa (including 97 of the 102 genera in the family) using the Angiosperms353 probe set. This pair of analyses produced highly concordant phylogenies and provides strong support for the clades included in the classification. In total, we propose and define rank-free names for 38 major clades, including 16 infrageneric taxa within the mega-diverse Solanum. This rank-free system intends to promote name stability as new data become available, avoiding name changes to well-known groups.

Extensive multi-species hybridization between Leuciscidae minnow species

Meuser, A. V. — 2025-07-18

Anthropogenic disturbances can disrupt ecosystems and alter species population dynamics. Interspecific hybridization is common between genetically related organisms, especially once reproductive barriers such as spatial isolation have been removed. We used genotyping- by-sequencing data to assess outcomes of hybridization between several Leuciscidae minnow species and to identify to what extent land use type and environmental variables influence the frequency of hybridization. We found that both two-species and multi-species hybridization was widespread; hybrids were sampled at all 25 sampling sites and made up almost 30% of all individuals sampled. While most species hybridized with at least one other sampled species, the amount of hybridization was variable. We used logistic regression to estimate the influence of anthropogenic disturbance on hybridization, and found significant but weak relationships between hybridization and environmental factors. This research improves our understanding of hybridization dynamics in species-rich clades like the Leuciscidae with low reproductive isolation, and points to the need for additional work to better understand predictors of hybridization in multi-species hybrid zones.

Sampling intensity and temporal persistence of airborne eDNA in partially enclosed spaces

Garrett, N. R. — 2025-07-18

Airborne environmental DNA (eDNA) has shown promise as a terrestrial biomonitoring tool and its ecological applications are expanding. Despite its growing use, airborne eDNA does not yet have the extensive body of supporting research like its aquatic counterpart, with considerable uncertainty remaining concerning how airborne eDNA behaves, with regards to signal duration, and how much sampling effort is needed to capture DNA in a given airspace. By using airborne eDNA in a semi-controlled environment which acted as an artificial roost where bat species and their abundances were known, we estimated the sampling intensity (both the number of samples and number of sampling events) required to capture bat diversity of a given airspace, as well as signal persistence of airborne eDNA. Together these data provide a temporal scale for airborne eDNA measurements. The majority of species richness was detected using as little as 4 samplers in this enclosed space and the greater the number of sampling events, the fewer samplers were needed. Both air movement and the type of environment (i.e., enclosed space, open area etc.) are likely to impact detection and need to be considered during study design. eDNA also appeared to settle out of the air quickly, suggesting that detections likely reflect recent activity, which also has important implication for rare species which may only have a narrow window for detection. Our results add to the growing body of literature that indicate airborne eDNA can be a useful biosurvey method, especially for rapid surveys in communities with high turnover rates.

Megabarcoding dark taxa - Assessing the utility of mass DNA barcoding for phorid fly species discovery

Vihavainen, J. — 2025-07-24

Many hyperdiverse, small-bodied insect families contain numerous undescribed species, generally termed "dark taxa". Scuttle flies (Diptera: Phoridae), being among the most diverse insect groups globally, are a prime example. DNA barcoding can help delineating dark taxa, particularly when integrated with morphology, and/or additional molecular evidence. We sequenced COI-barcodes from 9,120 Finnish phorid specimens and initially identified them using BOLD database. Furthermore, species identifications of all 843 non-Megaselia specimens were confirmed morphologically. Initially, the BOLD-based identifications matched the morphological identifications only in 68% of the cases, which resulted from many misidentifications in BOLD. After adjusting the BOLD reference identifications based on morphological analyses of male features, we established a reliable framework for female identification. This is advantageous for future identification of females, as they are often excluded from traditional identification keys. Only two species was discovered as new to Finland, demonstrating that Finnish non-Megaselia fauna is well-known. Although DNA barcodes show great promise for identifying phorids, incorrectly identified reference sequences remain challenging, not the functionality of COI itself. The number of Megaselia BINs greatly exceeded the known Finnish species count, with many sequences lacking matches in BOLD. This further highlights Megaselia as a particularly dark group, for which genetic tools are essential for uncovering species identities and assessing diversity.

Early-Life Poultry-Derived Lactobacilli Drive Microbial Succession and Gut Immune Modulation in Broiler Chickens

Abdelaziz, K. — 2025-08-02

Probiotic supplementation supports poultry gut health by modulating microbiome and promoting immune development, yet limited information is known about the effects of early, particularly embryonic, supplementation. In this study, we investigated the effects of administering a lactobacilli cocktail in ovo (embryonic day 18), post-hatch, or both on gut immunity and the succession of the cecal microbiota in broilers over five weeks. 16S rRNA gene-based sequencing of cecal contents revealed a steady increase in Shannon diversity during the first three weeks (PERMANOVA, p < 0.005), with community structure stabilizing by week 3 across all groups. In ovo lactobacilli administration improved early hatch rates and modulated microbial composition during early succession, including reductions in Klebsiella and Enterococcus, and enrichment of Lactobacillus, during the first two weeks (MaAsLin2, q < 0.25). These microbiome shifts were accompanied by a reduced expression of pro-inflammatory cytokines (IFN-{gamma}, IL-1{beta}, and IL-8) in cecal tonsils. These findings highlight the transient yet critical role of early-life probiotic interventions in shaping gut microbial colonization and immune response in broiler chickens. More importantly, a single in ovo lactobacilli dose yielded effects comparable to weekly oral or combined administration.

Early Subclinical and Sex-Specific Cardiac Remodeling Precedes HeartFailure in Zebrafish with Human Actin T126I Mutation.

Prill, K. — 2025-08-29

Abstract/SummaryDilated cardiomyopathy (DCM) is a leading cause of heart failure with notable sex differences in disease susceptibility and progression. Mutations in sarcomeric proteins such as cardiac actin (ACTC1) contribute to familial DCM, yet the in vivo consequences of the ACTC1 p.T126I variant and its sex-specific effects remain unclear. We generated a zebrafish model carrying the orthologous Acta1b p.T126I mutation and conducted longitudinal analyses of cardiac phenotype, function, morphology, and gene expression stratified by sex. Mutants showed variable onset of cardiac dysfunction; surviving adults developed progressive DCM characterized by pericardial effusion, ventricular dilation, and reduced survival. Female mutants exhibited earlier and sustained diastolic dysfunction, more severe cardiac remodeling and significantly lower survival compared to males, revealing pronounced sexual dimorphism. Molecular profiling identified upregulation of the cardiac stress marker nppb, downregulation of hypertrophic transcription factors (gata4, mef2ca), stable or reduced sarcomeric gene expression, and sex-specific alterations in calcium handling genes (serca2, pln1, slc8a1a) and proteostasis regulators (hsf1, bag3). These findings demonstrate that the Acta1b p.T126I thin filament mutation drives progressive, sex-specific dilated cardiomyopathy in zebrafish, underscoring biological sex as a critical modifier of sarcomeric cardiomyopathy progression and providing a valuable platform for investigating sex-dependent mechanisms and developing targeted therapies relevant to womens cardiovascular health.

The 'Iceberg region of the Grapevine leafroll-associated virus 3 replicase polyprotein contains signals for mitochondrial targeting and outer membrane association

Fust, C. — 2025-09-07

Positive-sense single-stranded (+ss) RNA viruses such as Grapevine leafroll-associated virus 3 (GLRaV3) replicate their genomes within membrane-bound viral replication complexes (VRCs). The biogenesis of such VRCs is driven by viral "replicase" polyproteins containing several replication-related domains. Past electron microscopy evidence suggests that GLRaV3 forms VRCs from the outer mitochondrial membrane of host grapevine plants. Here, we report the subcellular localization of the replicase polyprotein encoded by GLRaV3 ORF1a (PP1a) towards understanding its putative role in VRC formation. Through confocal laser scanning microscopy analysis of distinct EGFP-tagged PP1a domains, interdomain regions and truncations expressed in model plants, we report the molecular signals responsible for the targeting and association of PP1a to the mitochondria. This signal, located in the "Iceberg" region downstream from the methyltransferase-guanylyltransferase (M/GTase) domain, comprises an amphipathic -helix and a downstream transmembrane domain (TMD). Mutagenesis studies suggest that the polar face of the amphipathic -helix functions as the targeting signal, whereas the non-polar face, together with the TMD, act in membrane-anchoring. Microscopy observations are confirmed through mitochondrial isolation via gradient centrifugation and Western blotting. Structure prediction of the GLRaV3 M/GTase domain and its downstream TMD suggests a putative dodecameric oligomeric state. This dodecamer may gate the neck of GLRaV3 VRCs and contribute to their biogenesis, a hypothesis to be tested in follow-up studies. SIGNIFICANCE STATEMENTPositive-sense RNA viruses such as GLRaV3 form specialized membrane-bound viral replication complexes (VRCs) within host cells to sequester viral genome replication. Past evidence suggests that GLRaV3 targets the outer mitochondrial membrane (OMM) of grapevine host cells for VRC formation, however the viral protein responsible remained unknown. Here, we report a newly identified amphipathic -helix and a downstream transmembrane domain in the replicase polyprotein encoded by GLRaV3 ORF1a that are crucial for OMM targeting and membrane association. Since OMM targeting mechanisms in plants are poorly understood, our findings not only shed light on how GLRaV3 assembles VRCs, but also provides insight into OMM targeting more broadly. This work lays the foundation towards elucidating the molecular mechanisms of GLRaV3 replication and host-pathogen interactions.

Peracetic Acid Efficacy and Decay Kinetics in Poultry Processing under Chiller Conditions

Ciluveru, V. — 2025-09-08

Pathogens on poultry products continue to pose critical public health risks. Despite significant literature examining sanitizer impact on bacterial pathogens during chilling, the mechanisms of sanitizer efficacy in terms of pathogen and organic load, sanitizer levels, and exposure duration are not well understood. To assess these, we report on experimentally-informed-mechanistic-modeling to describe pathogen dynamics during poultry chilling. The shedding and survival of a five-strain cocktail of poultry-plant derived Salmonella enterica serovars, at high and low loads, with exposure to peracetic acid (PAA; 0 - 200 mg{middle dot}L-1) for up to 60 min in 10-L chiller tanks, in the presence or absence of whole chicken carcass/parts, were measured. Process water parameters versus time were simultaneously monitored. Results suggest that total dissolved solids (TDS) predict PAA decay more consistently than chemical oxygen demand (COD). A mathematical model for PAA decay and pathogen shedding/inactivation was developed. This model accurately predicted PAA level changes in the pre/main chiller of a high-speed poultry processing plant in North America. Without PAA, Salmonella shedding from chicken thighs is influenced by rinse time and number of rinses. Without organic load, residual PAA (1 mg{middle dot}L-1) inactivated bacteria given sufficient exposure time, although PAA levels > 5 mg{middle dot}L-1 were essential for rapid inactivation. With organic load, initial PAA concentration (> 40 mg{middle dot}L-1) and exposure time (> 2 min) were critical for bacterial inactivation, with model results connecting process conditions to dominate modes of bacterial inactivation on chicken. The insights from such experimental-modeling studies provide key tools for processors to improve pathogen control during chilling.

Cannflavin B ameliorates social and anxiety deficits and neuronal systems dysfunction in adolescent rats exposed to prenatal valproic acid

Williams, O. O. F. — 2025-09-17

There has been growing interest in natural products as potential therapeutics for the core and comorbid symptoms of autism spectrum disorders. Almost all the studies on autism have focused on the therapeutic benefits of cannabis and its associated cannabinoids. In this study the potential therapeutic efficacy of cannflavin B, a related, yet non-psychoactive component of the Cannabis sativa plant, was evaluated. Using prenatal valproic acid (VPA) exposure in rats, a model that has been widely used to study aspects of autism, we showed that cannflavin B was anxiolytic in the female VPA rats, and normalized sociality in VPA animals of both sexes. When neuronal oscillatory activity was examined, in female VPA rats cannflavin B normalized alterations in low frequency power within the cingulate cortex (Cg), and theta-gamma cross frequency coupling between the dorsal hippocampus (dHIP) and the prefrontal cortex (PFC). In male VPA animals, cannflavin B induced frequency-specific alterations in power within the PFC, Cg, and dHIP and ameliorated the VPA-induced suppression of oscillatory coherence between all three regions. In each brain region, cannflavin B also attenuated the sex-specific VPA-induced elevations in microglia. In vitro, cannflavin B normalized VPA-induced elevations in cortical and HIP neuronal activity and promoted more organized cortical firing. These findings demonstrate cannflavin B normalizes behavioural and neuronal systems function alterations induced by prenatal VPA in rats. The present study highlights the importance of alternative cannabis compounds in autism and other disorders.

Acute exogenous ketone monoester supplementation decreases cardiac vagal modulation in a dose-dependent manner

Thiessen, J. S. — 2025-09-21

Exogenous ketone supplementation can elevate heart rate (HR) but the effects on cardiac autonomic modulation remain unclear. HR variability was analyzed from 18 healthy adults (23 {+/-} 3 yrs; n=10 females) ingesting 0.3 (low-dose) or 0.6 g/kg (high-dose) ketone monoester or placebo in a randomized crossover, double-blind design. Compared to placebo, both ketone doses increased capillary {beta}-hydroxybutyrate and reduced the percentage of successive R-R intervals that differ by [≥]50-ms (pRR50) and the root mean square of successive differences (RMSSD) at 45- and 120-min post-ingestion (all p<0.05). Acute ketone ingestion decreases HR variability measures associated with reduced cardiac vagal modulation.

Assembly and substrate engagement mechanism of the bacterial proteasome activator Boa

Davis, B. T. V. — 2025-10-01

The bacterial proteasomal activator Bpa (Rv3780) is an ATP-independent regulatory particle of the Mycobacterium tuberculosis proteasome system. Bpa recruits substrates as a dodecamer and triggers the gate opening of the proteasome 20S core particle; however, the structural basis for its oligomerization and substrate recognition remains unclear. Here, we define the temperature-sensitive oligomerization mechanism of Bpa and elucidate its interaction with a non-native substrate. Using size-exclusion chromatography, charge detection mass spectrometry, and pulsed hydrogen/deuterium exchange mass spectrometry (HDX-MS), we show that Bpa reversibly assembles into a dodecameric ring from dimeric and tetrameric species in a temperature-dependent manner. We used HDX-MS to map the oligomerization interfaces during Bpa assembly. Methyl transverse relaxation optimized spectroscopy (TROSY)-based NMR experiments and site-specific truncations further validate the existence of discrete tetrameric and dodecameric states. To overcome the limitations posed by the poor solubility of the native substrates of Bpa, we establish the DNA-binding domain of hTRF1 as a surrogate substrate. Bpa binds hTRF1 and mediates its degradation in a 20S CP-dependent manner. We quantify the affinity and stoichiometry of the Bpa-hTRF1 interaction using methyl-TROSY NMR, identifying a 12 Bpa subunit : 3 hTRF1 binding ratio with micromolar affinity that is modulated by salt concentration. Our NMR-based mapping experiments pinpoint the interaction surfaces on both Bpa and hTRF1, revealing key hydrophobic residues that mediate substrate engagement. This work uncovers a thermosensitive switch regulating Bpa oligomerization and activity and introduces a tractable substrate for dissecting proteasomal recognition in M. tuberculosis.

Fish spawning events stimulate trophic hotspots across freshwater food webs

Fernandes, T. J. — 2025-10-02

1.0Mass reproductive events, where large numbers of organisms aggregate for synchronous reproduction, are often captivating and ecologically significant, boosting offspring survival and reducing time spent searching for mates. However, mass reproduction can also instigate cryptic but consequential responses across entire food webs. Reproductive materials are abundant and accessible resources that can attract mobile consumers from up to thousands of kilometers away. Yet, their consumption, especially in aquatic systems, is difficult to detect and rarely characterized. Here, we combine molecular techniques with acoustic telemetry, literature review, and extensive natural history observations to investigate the food web consequences of synchronized reproduction in freshwater fishes. First, we demonstrate that a common but underappreciated fish species, white sucker, creates a resource pulse used ubiquitously by consumers, from local invertebrates and fishes to mobile predatory fishes, birds, and terrestrial mammals. Spawning white sucker create trophic hotspots that attract consumers across trophic levels and ecosystems to feed on eggs, spawning adults, and aggregated egg predators. Then we show that egg provisioning and predation is widespread among north-temperate freshwater fish species, highlighting that resource pulses instigated by mass reproduction may play a critical but underappreciated role in freshwater and terrestrial ecosystems.

beta3-adrenergic browning of pericardial adipose tissue controls cardiac function

Brunetta, H. — 2025-10-02

Dysfunctional adipose tissue (AT) is strongly linked to the development of cardiovascular diseases (CVD). Accumulation of AT around vital organs is detrimental to their respective function and overall health. Although there is strong evidence linking the accumulation of pericardial AT with CVD development, a comprehensive investigation on the adaptation of pAT in obesity is scarce. Here, by applying pair-wise bottom-up proteomics in pAT of humans and mice, we found pAT presents a browning signature, as demonstrated by enrichment of mitochondria, presence of UCP1, and greater metabolic capacity compared to subcutaneous AT. In mice fed a high-fat diet or obese patients, the pAT undergoes whitening, characterized by adipocyte hypertrophy, reduced mitochondrial content, respiratory capacity, and UCP1 levels. Lipectomy of pAT from obese mice decreased pathological ventricular hypertrophy. Conversely, selective {beta}3-adrenergic agonist treatment rescued pAT browning status and is associated with improved heart structure and function, including ventricular thickness, and fibrosis in obese mice. Importantly, lipectomy of pAT abrogated the positive effects of {beta}3-adrenergic agonism in cardiac function of obese mice. Altogether, our work positions pAT as a mechanistic driver of obesity-related cardiac dysfunction and establish {beta}3-adrenergic-mediated browning of pAT as a novel therapeutic treatment strategy.

Convergent evolution of aerobic fermentation through divergent mechanisms acting on key shared glycolytic genes

Horianopoulos, L. — 2025-10-02

As the tree of life becomes increasingly accessible to molecular investigations, describing mechanisms underlying evolutionary convergence and constraint will be crucial to understanding diversification. The lineage including the model yeast Saccharomyces cerevisiae evolved aerobic fermentation in part through an ancient whole genome duplication and retention of glycolytic genes. To evaluate the glycolytic rates across diverse yeasts, we developed and deployed an extracellular acidification rates (ECAR) assay on 299 species that span more than 400 million years of evolution and identified a clade in the genus Saturnispora that convergently evolved aerobic fermentation. Through comparative genomics and transcriptomics, we found that several glycolytic genes had higher expression and novel cis-regulatory elements in aerobically fermenting Saturnispora species. When the transcription factor required for their activation was deleted in Saturnispora dispora, the mutants had reduced glycolytic rates and increased respiration. Intriguingly, many of the upregulated genes are orthologous to duplicated glycolytic genes in S. cerevisiae. These divergent genetic mechanisms suggest that there are strong evolutionary constraints on how some traits like aerobic fermentation can arise convergently.

Hemifield-Specific Motion Extrapolation Reveals Limits of Interhemispheric Integration

Olenick, C. E. — 2025-10-07

The accurate perception of moving objects is a fundamental challenge for the visual system, which must compensate for neural processing delays. Motion extrapolation is a proposed mechanism whereby the brain predicts an objects future position. We investigated how an objects motion history shapes its perceived position using the flash-jump illusion, in which a brief color change in a moving bar is mislocalized as further along the direction of motion. Across two experiments, we found that longer preceding motion sequences improved localization accuracy, consistent with motion adaptation. This effect occurred regardless of whether motion continued after the flash. Notably, mislocalization transiently reappeared as the object crossed the vertical midline, suggesting that motion adaptation and motion extrapolation operate independently between within each hemifield. Manipulating the length of the sequence in each hemifield in Experiment 2 confirmed that this adaptation is spatially confined to each hemifield, with limited interhemispheric transfer. The results align with a Bayesian framework in which the brain integrates signals from both hemispheres, with midline crossings triggering a shift from adapted to unadapted neural populations. We identify motion extrapolation, supported by hemifield-specific adaptation in area MT and integration in area MST, as the mechanism behind these midline discontinuities. This work reframes smooth pursuit not just as a tracking behaviour, but as a functional solution to overcome the inherent limits of interhemispheric motion processing.

Positive selection on hotspot and reinforcing regulatory alleles contributed to hexaploid bread wheat improvement.

Zhan, S. — 2025-10-08

BackgroundGenetic variation of regulatory alleles plays a key role in evolution and breeding. In polyploids, regulatory differences may preferentially affect genes on homoeologous chromosomes or sub-genomes. Selection in plant breeding may act upon total transcript dosage across homoeologous genes and on alleles that have strong effects on the transcriptome. ResultsTo investigate these questions, we identified regulatory polymorphisms between an old and a recent hexaploid bread wheat cultivar (Triticum aestivum, 2n=6x=42, AABBDD). The recent cultivar was the product of decades of selection for grain yield and quality. Regulatory allele polymorphisms preferentially affected genes on homoeologous chromosomes but rarely affected genes on specific sub-genomes. The chromosomal distributions of regulatory alleles indicated that past selection had acted upon them, and the effect of selection differed between alleles targeting environmental response genes and genes involved in other processes. Modern cultivar alleles that affected many genes transcripts corresponded to known selection targets and improved field crop performance. Modern cultivar alleles also had significant effects on homoeologous genes, and these alleles also improved crop performance. ConclusionsPolyploid breeding across many species has been and will continue to be the key factor in plant improvement. By enhancing the favorability of strong regulatory alleles and by expanding the range of gene transcript abundances, genome duplications enable breeding progress.

Lipid acyl chain length and unsaturation modulate membrane surface charge and interactions with amphiphilic DNA nanoprobes

Li, Y. — 2025-10-13

Many cellular pathways rely on the active regulation of membrane composition to fine-tune key physico-chemical properties, including lipid packing, fluidity, and surface charge. While intrinsic membrane charge is often attributed to specific lipid headgroups and membrane-bound proteins, the contribution of acyl-chain chemistry to the electrostatic profile of membrane surfaces remains unexplored. Here, we systematically investigate how variations in acyl chain length and unsaturation modulate the biophysical and electrostatic properties of zwitterionic lipid membranes. Using amphiphilic DNA nanoprobes as model charged biomolecules, we reveal how lipid packing, fluidity, and membrane phase collectively govern surface charge and interactions with nanoprobes, delineating relationships that persist in the presence of anionic lipids. We further demonstrate that the identity and hydrophobicity of membrane anchors in nanoprobes significantly influence binding, providing a means to modulate membrane association through programmable strategies. By establishing design principles that link acyl chain chemistry to surface charge and biomolecular attachment, our findings provide a mechanistic framework to engineer selective membrane interactions. Beyond their direct applicability to biomimetic platforms and synthetic cell engineering, these insights hold broad relevance to lipid-based vaccine nanotechnologies and the fundamental understanding of membrane-biomolecule interactions in living cells.

HyperdCas12a-Based Multiplexed Genetic Regulation in Candida albicans

Gervais, N. C. — 2025-10-20

Complex microbial phenotypes involve the combined activity of diverse gene regulatory networks. However, the majority of reverse genetics approaches in microbial pathogenesis research have focused on single-gene perturbation studies, in part due to the lack of available genetic tools in many pathogens. Developing enhanced versions of CRISPR-Cas platforms holds significant promise for improving the scalability of microbial functional genomics research. Here, we demonstrate highly efficient, inducible, and multiplexed activation and repression in the major human fungal pathogen Candida albicans by translating the hyperdCas12a variant to the fungal kingdom. This represents the first application of a CRISPR-Cas12 system in a human fungal pathogen. We profile the effectiveness of our new CRISPRa and CRISPRi tools and achieve tunable levels of target modulation. Further, we demonstrate that perturbing combinations of genes in the drug efflux and ergosterol biosynthesis pathways reveals important redundancies and synergistic properties in drug resistance circuitry. Our hyperdCas12a platform is thus an efficient system for the rapid generation of combinatorial mutants that will enable the mechanistic understanding of genetic interactions involved in diverse phenotypes in C. albicans. The enhanced activity with hyperdCas12a in fungi suggests it could be translated to other microbes as a powerful tool for studying genetic interactions.

Visual Attention in Peripersonal Space

Ramezanpour, H. — 2025-10-21

Bringing the hand near a visual stimulus enhances visual processing. This effect is linked to peripersonal space (PPS), the body-centered region where visual and proprioceptive information interact. Despite extensive behavioral evidence, the neural basis of this interaction in early visual cortex remains unclear. In this study, we investigated how hand proximity modulates orientation selectivity in area V2 by recording single-neuron responses from two rhesus monkeys. The monkeys performed a fixation task while their hand was positioned near a visual stimulus while either being visible or occluded, and compared with when the hand was away from the stimulus. When the near hand was visible, neural firing rates in V2 were significantly higher, accompanied by sharper orientation tuning. In contrast, occluding the hand broadened orientation tuning compared to when the hand was away. These effects emerged rapidly after stimulus onset and were coherent across the population, demonstrating that PPS is actively prioritized during visual processing. Together, the findings reveal two complementary (feedback) signals in V2: a congruence-driven enhancement when visual and proprioceptive inputs align, and a mismatch-driven suppression when they conflict, indicating that V2 integrates multisensory cues to encode PPS and support action-relevant visual processing.

STING activation reshapes the tumor microenvironment leading to tumor regression in osteosarcoma

Young, E. P. — 2025-10-25

Osteosarcomas are characterized by a high degree of aneuploidy, chromothripsis and micronuclei, yet these tumors typically have an immunosuppressive, macrophage-rich, T-cell depleted tumor microenvironment. cGAS-STING dysregulation is a possible mechanism by which immune activation in response to tumor genomic instability could be repressed. We identified almost universal repression of cGAS or STING in human osteosarcomas. However, a STING-activation gene signature was predictive of survival in osteosarcoma patients suggesting potential for activation of this pathway in the osteosarcoma tumor microenvironment. Indeed, in immunocompetent osteosarcoma models, systemic STING agonism led to complete regression and induced lasting immunologic memory. Host STING activation is sufficient to promote this anti-tumor immunity even in the absence of tumor STING. These results nominate the cGAS-STING pathway as an important therapeutic target in osteosarcoma, a disease in which no new curative therapies have been developed in the last 40 years. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/684275v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@15b1e5corg.highwire.dtl.DTLVardef@12650aaorg.highwire.dtl.DTLVardef@79ef71org.highwire.dtl.DTLVardef@15704c9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Transient dynamics and counterintuitive competitive performance in periodic environments

Bieg, C. — 2025-10-27

Despite the rapid pace of global change altering temporal environmental patterning, we lack a general understanding of how periodic environments structure ecological communities. In fluctuating environments, nonlinear dynamics associated with temporal trade-offs between competing species can create the potential for both niche differentiation (coexistence) and seemingly unexpected outcomes (exclusion) that deviate from deterministic coexistence theory. Yet, the mechanisms behind these outcomes are not fully understood. Here, we show that periodic fluctuations between times of high and low growth (e.g., seasons), and adaptive temporal trade-offs within and between species, can drive counterintuitive over- and under-performance of competing species. Most notable is the counterintuitive outcome of seasonally-mediated competitive exclusion that would not occur in either season alone, but is rather the direct result of environmental variability itself. We find that seasonal trade-offs in species growth rates, seasonal differences in competition strength, and functional similarity between competing species have the potential to drive nonlinear responses in coexistence to changing seasonality under global change. These biological conditions collectively influence our models transient dynamics, further explaining the mechanisms behind counterintuitive outcomes and highlighting the importance of non-equilibrium theory for global change ecology. Importantly, the seasonal patterns and species trade-offs that magnify these results are biologically realistic, therefore providing important insight into the implications for the maintenance of biodiversity under global change.

Species interactions, divergence, and the rapid evolution of ecological sexual dimorphism in threespine sticklebacks

Blain, S. — 2025-10-27

Variation in ecological sexual dimorphism (ESD), defined as differences between the sexes in ecologically-relevant traits, is a common feature of adaptive radiation, yet its causes remain unclear. Competition between the sexes for alternative resources can promote evolution of ESD when interspecific competition is reduced (competition hypothesis). Alternatively, sex-specific selection on ecological traits might weaken under strong directional selection in new environments (divergence hypothesis). We tested these hypotheses via their expected evolutionary outcomes in threespine stickleback populations from southwestern Canada. We found striking among-population variation in magnitude of ESD. Consistent with the divergence hypothesis, dimorphism along the main axis of body shape variation was reduced in recently derived freshwater populations compared to their contemporary marine ancestor. However, dimorphism only weakly declined with increasing phenotypic divergence. Average dimorphism along major freshwater body shape axes was similar between solitary populations and those coexisting with a competing species, contrary to the competition hypothesis. Instead, sympatry with a Benthic ecotype led to increased sexual size dimorphism in Limnetics, and total shape dimorphism was elevated in the sympatric stickleback species compared with solitary populations. In contrast to the mechanisms considered in existing theory, interactions in sympatry might produce elevated ESD by generating novel sex-specific selection pressures.

A trait syndrome ties cell morphology to glycolysis across the yeast subphylum

Horianopoulos, L. C. — 2025-10-30

Traits that co-vary across species can provide fundamental insights into the trade-offs and constraints that govern their evolution. It was recently reported that glucose uptake rates (GUR) are inversely correlated with the cell surface area-to-volume (SA:V) ratio across 11 yeast species. Here we substantially expand this analysis to 282 species to test whether the GUR-SA:V correlation generalizes across the ancient Saccharomycotina yeast subphylum and to determine the contribution of shared evolutionary history to the co-variation of these two traits. Using regression models that account for co-variation due to phylogeny, we found that extracellular acidification rates (ECAR, which we used as a scalable proxy for GUR) had a weak, but significant, correlation with SA:V across Saccharomycotina. We found additional weak, but significant, correlations between ECAR with genome sizes and growth rates. Our findings largely agree with the recently reported correlations between GUR and SA:V ratio, but they also show that there are likely several other associated variables, including genome size. Specifically, yeasts that consume glucose faster tend to have lower SA:V, faster growth rates, and larger genomes. Our results suggest that a trait syndrome governs several metabolic, genomic, and morphological traits across the yeast subphylum.

Ecological coherence in abundance dynamics across terrestrial and marine assemblages

Fuster-Calvo, A. — 2025-11-05

Understanding how communities respond to environmental change requires assessing not just overall variability but also the structure of co-variation among taxa. We frame this idea under the Ecological Coherence (EC) framework, which generalizes previous notions such as community synchrony or coherence. EC captures the structure of co-responses among taxa within assemblages and can be expressed through two complementary objects: (1) the co-response matrix C, which contains all pairwise correlations between taxa and can be used to identify clusters of taxa with coherent responses as well as the contributions of individual species to community-wide coherence; and (2) the EC distribution, which summarizes the overall profile of co-responses by capturing their shape, spread, and central tendency across the community. By combining these two views, EC moves beyond single summary metrics and provides a richer picture of how coherence is organized within communities. Analyzing the EC distribution across 341 terrestrial and 105 marine assemblages worldwide, we found a general prevalence of weak correlations and a few strong, directional correlations. We also found that it varies with community composition, sampling effort, and biogeographic region. Moreover, the C matrix consistently identified a small subset of taxa with strong correlations to many others, suggesting a promising path to detecting those that may play central roles in amplifying or buffering community responses to environmental change. Our findings on EC pave the way for deeper investigations into what drives the diversity of ecological responses to environmental changes and how it shapes community dynamics, while also underscoring the need for strategically distributing ecological monitoring across trophic guilds and functional roles.

The Unique Efg1 Fungal Virulence Regulon in the Catheterized Bladder Environment

La Bella, A. A. — 2025-11-07

Urinary catheterization, a common procedure in hospitals and nursing home facilities, is a primary driver of hospital-acquired infections (HAI). These devices frequently lead to catheter-associated urinary tract infections (CAUTIs), which often progress to severe complication, sepsis, and ultimately death. The fungus Candida albicans has emerged as the second most common causative agent of CAUTIs; yet, its pathogenesis is poorly understood, which complicates development of efficient treatments. Previously, we identified the transcription factor Efg1 as a critical virulence driver in C. albicans CAUTIs. However, its specific downstream targets within the unique bladder microenvironment remained unknown. This study identifies, for the first time, the complete Efg1 regulon that is active during growth in human urine. We confirmed the clinical relevance of this discovery, finding that many of these Efg1-regulated factors are present and significantly upregulated in catheter samples from patients with C. albicans infections. Furthermore, we characterized two of these key factors, ECE1 and EED1, validating their roles both in vitro in urine conditions and in vivo using a CAUTI mouse model. Identifying the tissue-specific downstream targets of Efg1 elucidates the precise mechanism of fungal CAUTI. This knowledge provides a new roadmap for developing targeted therapeutics, offering vital antimicrobial-sparing strategies to combat these life-threatening infections. SIGNIFICANCECatheter-associated urinary tract infections (CAUTIs) are common hospital-acquired infections that can lead to severe complications and death. Although most are caused by bacteria, the fungus Candida albicans is an increasingly prevalent cause, yet the pathogenesis of fungal CAUTIs is poorly understood. Previous research identified Efg1 as necessary for CAUTI, and now this study defines the urine-specific Efg1 regulon, validating its clinical relevance in catheter samples from infected patients. We further assessed how key downstream factors, Ece1 and Eed1, contribute to bladder infection. This first report of the urine-specific EFG1 network provides new targets for diagnosing and treating these life-threatening infections.

Is there a fly in my soup? To what extent do metabarcoding and individual barcoding tell the same story?

Furneaux, B. — 2025-11-13

O_LIMetabarcoding has become the method of choice for characterising complex arthropod communities. The extent to which metabarcoded bulk samples will recover the same community composition as individual sequencing of all individuals in the sample remains poorly quantified. Biases such as unequal extraction of DNA from different taxa, primer mismatches and non-random PCR may cause the selective drop-out of species from metabarcoding data. At the same time, DNA metabarcoding may reveal arthropod taxa present not as individuals, but as DNA residues on the surface or in the gut of insects. C_LIO_LITo quantify the consistency in sample contents established by different means, we metabarcoded 45 bulk insect samples, then extracted all arthropods and sequenced them individually. Metabarcoding targeted 418 bp at the 3 end of the Folmer barcoding region, while individual barcodes captured the entire 658 bp Folmer region. The metabarcoding workflow, including PCR amplification, sequencing, and bioinformatics, was performed in three replicates from three separate lysate aliquots per sample. Sequences were assigned to Barcode Index Numbers (BINs) as identical taxonomic categories across data types. C_LIO_LIWe found that metabarcoding is replicable, as different replicates of the same sample recover similar species richness and composition. Individual barcoding and metabarcoding provide the same impression of relative differences in community structure: estimates of relative species richness and relative dissimilarity between sample pairs are congruent among data types. Dissimilarity between data types varies with BIN richness in the sample, but this relationship reflects nestedness rather than turnover: metabarcoding recovers the same set of core species as individual barcoding but adds hundreds of species on top. Any BIN recovered as an individual occurred with high probability in the metabarcoding data, and any BIN found in high read abundances by metabarcoding was likely found as an individual. C_LIO_LIOur analysis suggests that metabarcoding data will closely mimic the sample contents in terms of arthropod species richness and composition. Taxa recovered in low copy numbers in metabarcoding sequence data will typically represent DNA left as residues from past biotic interactions. Barring sequencing errors, both types of data yield biologically relevant insights into the taxa present in the source community. C_LI

Oxidative-stress related increase in keratoconus tear MDA and GPX3 while NRF2-antioxidant functions decrease in stromal cells

Koduri, M. A. — 2025-11-14

Keratoconus (KC) is a common eye disease where the cornea undergoes degenerative thinning and steepening. The absence of biomarkers for early diagnosis prior to the onset of overt corneal phenotypes and the lack of curative treatments rooted in a fundamental understanding of KC biology remain significant challenges. To address these issues, we investigated the role of unresolved oxidative stress in KC pathogenesis. Malondialdehyde (MDA) a lipid peroxidation byproduct that accumulates during oxidative stress was significantly elevated in the tears of KC patients compared to unaffected controls and positively correlated with maximal keratometry (Kmax), a measure of KC severity. Similarly, the secreted antioxidant glutathione peroxidase 3 (GPX3), was significantly increased in patient tears, and strongly correlated with Kmax. In a cell culture model of oxidative stress, KC corneal stromal cells displayed increased apoptosis and suboptimal activation of NRF2, a transcription factor master regulator of antioxidant genes. Conversely, inhibition of NRF2 in donor stromal cells elicited KC-like cellular phenotype, whereas sulforaphane, an NRF2 booster restored antioxidant gene expression and the deposition of cornea-typical collagens. Our study identified cellular antioxidant signaling dysregulations in keratoconus where sulforaphane treatment may be restorative. Consistent increases in patient tear MDA and GPX3 present these as promising biomarkers for KC diagnosis and severity predictions.

Distinct Ire1-driven transcriptional responses control morphogenesis in Candida albicans

Stack-Couture, S. — 2025-11-14

The pathogenic yeast Candida albicans relies on morphogenesis--the transition from spherical yeast to filamentous hyphal forms--for infection. While morphogenesis requires Ire1, a transmembrane protein that canonically initiates the Unfolded Protein Response (UPR) through HAC1 mRNA splicing, the specific mechanisms linking Ire1 to filamentation remain unclear. Using transcriptome analysis, we found that the Ire1-dependent transcriptional response driving morphogenesis is fundamentally distinct from the canonical UPR response to proteotoxic stress, with minimal overlap between programs. Remarkably, morphogenesis occurs without detectable HAC1 splicing, and HAC1 deletion only partially impairs filamentation, unlike complete loss with IRE1 deletion. These findings establish that Ire1 regulates hyphal development through previously uncharacterized HAC1-independent pathways. Our data reveal decreased transcription of secretory proteins in an Ire1-dependent manner, providing compelling evidence that C. albicans possesses regulated Ire1-dependent decay (RIDD) activity--a post-transcriptional mechanism not previously characterized in this pathogen. Additionally, we identify cell wall integrity as a key HAC1-independent mechanism, with Ire1--but not Hac1--essential for cell wall stress tolerance and upregulation of cell wall biosynthesis genes during filamentation. Given Ire1s essential role in pathogenesis and extensive development of Ire1-targeting compounds for mammalian systems, our findings position Ire1 as a highly promising druggable target for novel antifungal therapeutics and development of fungal-specific inhibitors.

DHA Increases Angptl4 Gene Expression and Reduces LPL Activity in a PPARγ-dependent manner in Adipocytes

McTavish, P. V. — 2025-11-16

Omega-3 polyunsaturated fatty acids (N-3 PUFA), specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are well recognized for their triacylglycerol (TAG)-lowering properties. These effects are generally attributed to reduced hepatic lipogenesis and increased {beta}-oxidation; however, the contribution of white adipose tissue (WAT) towards the hypotriglyceridemic properties of N-3 PUFA is less defined. Lipoprotein lipase (LPL) regulates TAG hydrolysis to influence fatty acid uptake into WAT, a process that can be inhibited by angiopoietin-like 4 (ANGPTL4). When re-examining a previous mouse study, we found that mice consuming a diet rich in EPA/DHA had increased WAT Angptl4 expression in the fasted state compared to a control diet. Therefore, the goal of this study was to explore the role of N-3 PUFA on the regulation of Angptl4 expression and LPL activity in mouse adipocytes. 3T3-L1 adipocytes treated with DHA (100M), but not ALA or EPA, increased Angptl4 expression and reduced LPL activity similar to that observed with a PPAR{gamma} agonist (pioglitazone). When Ppar{gamma} expression was knocked down with siRNA, the ability of DHA and pioglitazone to induce Angptl4 expression was ablated. Further, DHA- and pioglitazone-induced reductions in LPL activity were mitigated when Angptl4 expression was silenced. Taken together, these results suggest that DHA regulates LPL activity by increasing Angptl4 expression in a PPAR{gamma}-dependent manner. Our results have uncovered a novel mechanism by which DHA regulates ANGPTL4 to influence LPL-mediated hydrolysis of circulating TAG in adipocytes. Future in-vivo studies are necessary to determine the relevance of DHA regulation of ANGPTL4 towards whole-body lipid homeostasis and cardiometabolic health. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/685896v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@75505dorg.highwire.dtl.DTLVardef@1ef29b4org.highwire.dtl.DTLVardef@9aec7dorg.highwire.dtl.DTLVardef@11c372b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDHA increases Angptl4 gene expression and reduces LPL activity in 3T3-L1 adipocytes C_LIO_LIChanges in FOXO1 do not explain DHA-induced changes in Angptl4 gene expression C_LIO_LISilencing Ppar{gamma} ablates DHA-induced increases in Angptl4 gene expression C_LIO_LISilencing Angptl4 partially attenuates DHA-induced reductions in LPL activity C_LI

Direct Measure of DNA Damage in Huntington Disease Reveals Elevated Oxidative Genotoxic Stress and Dysfunctional DNA Repair

Bazan, C. A. B. — 2025-11-23

DNA damage repair (DDR) pathway proteins are genetic modifiers of Huntington disease (HD) age of onset and severity. Deficient DDR in HD at the first steps of poly ADP-ribosylation makes DNA damage quantification by traditional downstream DNA damage response markers inaccurate. Repair Assisted Damage Detection (RADD) allows for an accurate direct assessment of oxidative DNA damage, a DDR pathway in which huntingtin protein directly participates. Using RADD, we show that human HD-derived cells have elevated oxidative DNA damage and assess the effect of relevant HD therapeutic targets in rescuing this phenotype. Using huntingtin protein level lowering, we define a dysfunctional role of mutant huntingtin in oxidative DDR. We show that ataxia-telangiectasia mutated (ATM) signaling is deficient and that ATM inhibition rescues elevated oxidative DNA damage in HD cells. In contrast, we show that N6-furfuryladenine (N6FFA) treatment, to increase huntingtin phosphorylation within the amino terminal N17 domain (p-N17), is not effective at restoring HD DDR but reveals dysfunctional N6FFA mediated DDR signaling. We propose a model in which elevated DNA damage arises from both aberrant mutant huntingtin involvement in oxidative DDR and the impairment of oxidative DDR pathways, such as ATM kinase activity, N6FFA processing, and poly ADP-ribose signaling in HD. This highlights the importance of using direct measures of DNA damage such as RADD, rather than measures of a DNA damage response pathway.

Fine-scale ecological biomonitoring in a large, complex agriculturally impacted watershed via eDNA metabarcoding

Silva, B. S. M. L. e. — 2025-11-27

DNA-based approaches utilizing high-throughput sequencing (HTS) (e.g. DNA metabarcoding) have revolutionized ecological biomonitoring by providing higher sample throughput, greater reproducibility, and better cost-benefits compared to traditional morphology-based bioassessment studies. Here, we utilized DNA metabarcoding in a watershed in Ontario (Canada) dominated by agricultural land uses. Our aim is to understand patterns of biodiversity in benthic taxa from data generated and inferred at various taxonomic scales and to compare these findings with over a decade of traditional morphological data. We sampled 18 watercourses during summer and fall 2023, spanning a forested-to-agricultural land-use gradient. We found significant differences between metabarcoding and historical morphology data where DNA provided more richness values at both the species (p = 2x10-5) and order (p = 0.008) levels. Whereas the morphology dataset contained many unresolved taxa, DNA metabarcoding captured a broader taxonomic breadth with diverse genetic profiles among taxa. Non-metric multidimensional scaling (NMDS) analyses on DNA metabarcoding data produced tighter clusters, more precise separation by land use, and greater consistency across taxonomic scales. Both urban context and land use had significant associations with metabarcoding patterns observed, with differences being strongest between agriculturally-dominated and primarily forested sites (median R{superscript 2} {approx} 0.08-0.11). We also found strong, consistent environmental signals linked to agricultural settings, such as water conductivity and turbidity, and pH. Altogether, our DNA-based results demonstrate the differences in community composition among different land uses in this watershed. Importantly, our work highlights the need for more taxonomic resolution (obtained through DNA analysis) to decipher community changes linked to anthropogenic and environmental drivers, as morphological data alone may lack the precision needed to capture these patterns.

Using GPT-4 to Automate the Generation of Lay Summaries for Cancer Publications

Purdie, E. — 2025-12-12

BackgroundCancer research literature is often riddled with technical jargon that is not digestible to the average person. Individuals interested in research studies may want to contribute through patient partner engagement or sample donation but find the relevant literature overwhelming. Through the generation of lay summaries, previously inaccessible research papers become easier to comprehend, especially for patient partners or data donors. With large language models (LLMs) continuing to advance, so does their capability to summarize large texts. ObjectivesIn this study, we examined whether LLMs can produce lay summaries of scientific literature at-scale, while maintaining readability and accuracy to their source texts. MethodsWe developed a tool to generate lay summaries of open-access article abstracts and their full texts with GPT-4-Turbo. Prompt development aimed for a target 8th grade reading level assessed with Flesch-Kincaid Grade Level. Human-review metrics were used to evaluate readability and accuracy when generated using abstracts versus full text articles. ResultsThe average Flesch-Kincaid Grade Level Score was 7.13 for abstract-based summaries and 7.39 for full text-based summaries, indicating summaries at around 7th grade reading level. Human-review metrics showed these summaries were of similar readability and accuracy when generated using abstracts versus full text articles, with mean accuracy scores from human review of 7.09 vs 7.42 out of 10 respectively. Additionally, qualitative patient-based assessment indicated these summaries would encourage participation in research studies. ConclusionBy generating lay summaries for complex and lengthy research papers, their scientific information becomes accessible to a larger audience, including patient partners interested in contributing to cancer research. Summaries that are easy to understand will allow participants to make informed decisions about their involvement and appreciate the impact of their contributions if and when their results are published. Lay SummaryThis study explores if artificial intelligence (AI) can help make hard to read cancer research papers easier to understand for members of the public. ProblemWhen people donate cancer tissue samples or participate in research studies, they often want to know how their contributions are being used. However, scientific papers are full of technical language thats hard for most people to grasp. People in past studies have said this can make them less willing to take part in research. MethodsThe study created a computer program using AI (GPT-4-Turbo) to turn complex kidney cancer research papers into simple summaries. They tested whether the AI could summarize both short abstracts and full-length papers effectively. They aimed for summaries at a 6th to 8th-grade reading level. This was to follow Canadian and U.S. health communication guidelines. ResultsThe AI created 106 summaries. Computer measures showed the summaries were close to a 7th-grade reading level. Though, researchers had to tell the AI to write for a 2nd-grade audience to achieve this. Of note, summaries from short abstracts were just as accurate and readable as those from full papers. Eighteen volunteers, including five patient partners, reviewed the summaries and rated them for clarity and accuracy. They were rated at around 7 out of 10 points. All patient partners said these summaries would help them decide whether to join research studies and feel more informed about how their contributions matter. Why It MattersThis tool could help patients and donors better understand research without needing a science degree. When people can see how studies work, they are more likely to participate in future research. While patient partners emphasized the need for summaries to be accurate and reliable, this approach shows promise as a unique strategy to better connect the public with research.

Enterocloster citroniae and related gut microbiome species modulate Vibrio cholerae biofilm formation through the production of bioactive small molecules

Pauer, H. — 2025-12-16

Cholera is a diarrheal disease that affects millions of people globally. Although the causative agent, Vibrio cholerae, has been extensively studied in isolation, investigation of its interactions with the gut microbiota started relatively recently. We and others previously showed that microbiota-derived metabolites significantly influence V. cholerae behavior. By investigating how an organic extract of human feces affects V. cholerae gene expression, we showed that gut metabolites strongly suppress swimming motility, a trait important for host colonization. Interestingly, extracts of pure cultures of a gut commensal, Enterocloster citroniae, recapitulated this inhibition. Here, we present a comprehensive examination of the effect of small molecules produced by E. citroniae and related species on V. cholerae behavior. We show that E. citroniae small molecules inhibit motility by various V. cholerae strains, and that several phylogenetically related species produce this activity, although the magnitude of the effect varies between strains. Using biofilm formation assays in static and flow conditions, we show that V. cholerae strongly induces biofilm formation in response to E. citroniae metabolites. Transcriptome and reporter analyses showed that several genes involved in synthesis of an extracellular polysaccharide are induced by E. citroniae metabolites. Lastly, we show that V. cholerae interactions with host cells are also modulated by this commensal. These findings advance our understanding of microbiome-pathogen interactions and how commensal bacteria influence V. cholerae virulence through the production of small molecules. In the future, this knowledge may be used to design novel microbiome-based therapeutic approaches to combat cholera and other infections. ImportanceThe human gut is home to a dense and rich community of microbes termed microbiota. This community has critical functions for host health, including protection against enteric pathogens. Despite this important role, we have only recently scratched the surface of the interactions that occur between members of the microbiota and pathogenic invaders. Cholerae is a disease that still causes significant morbidity and mortality worldwide. Studying how the causative agent, Vibrio cholerae, interacts with the microbiota will have implications not only for our understanding of this important microbial community, but may also lead to the development of new therapeutic strategies against cholera and potentially other infectious diseases.

Pooled CRISPRi screening reveals fungal-specific vulnerabilities across environments and genetic backgrounds

Wensing, L. — 2025-12-22

The rising rate of drug-resistant fungal infections and the emergence of fungal pathogens with intrinsic resistance phenotypes are a growing concern. The close evolutionary distance between mammals and fungi complicates the design of new antifungals and increases the chances of toxic off-target effects. As such, antifungal drug development usually focuses on fungal-specific proteins when considering potential new targets. Ideal drug targets should mediate essential cell processes and be highly sensitive to inhibition. Targeted gene repression can serve as a model for drug-mediated inhibition and for determining the dosage-sensitivity profile of genes of interest. In the fungal pathogen Candida albicans, classical approaches for gene repression can be labour-intensive and limited to one genetic background due to low throughput. Here, we adapt pooled CRISPRi screening in C. albicans for the first time and exploit this technique for large-scale functional genomic analysis. Through pooled CRISPRi screening, we test the repression sensitivity of over a hundred essential genes conserved in fungi but absent in humans, and successfully identify highly dosage-sensitive genes across multiple cell components and pathways. By extending our analysis to ten diverse environmental conditions, we show how the environment influences dosage-sensitivity profiles. Finally, we extend our experiments to two clinical drug-resistant C. albicans strain backgrounds and demonstrate that many of the fitness defects we observed are conserved in resistant clinical isolates. Together, our results highlight a set of genes that are highly dosage-sensitive across different genetic and environmental contexts, making them attractive targets for further investigation. By facilitating rapid, efficient large-scale functional genomics assays across diverse genetic backgrounds, CRISPRi pooled screening will open new frontiers in C. albicans biology.

RAMBO: Resolving Amplicons in Mixed Samples for Accurate DNA Barcoding with Oxford Nanopore

Kolter, A. — 2025-12-29

DNA barcoding, the use of short genetic markers to identify and differentiate species, is a foundational tool for ecological and taxonomic research. The method has been scaled rapidly with next-generation sequencing technologies enabling the processing of thousands of specimens in parallel. Nanopore sequencing not only offers a flexible, low cost alternative to other platforms but produces full-length reads in real time and can be used in remote settings. However, its comparatively high error rate complicates downstream processing, particularly when PCR amplifies multiple templates from a single specimen, reflecting pseudogenes, paralogs, or contaminants. We present a novel pipeline for DNA barcoding that resolves mixed sequence signals from Nanopore reads using unsupervised clustering and staged consensus generation, without relying on curated reference databases, taxonomic priors, or error models. While existing methods to curate Nanopore sequence data assume a single dominant amplicon per sample or require deep sequence divergence among amplicons, our pipeline can distinguish variants differing by as little as 0.15 percent. It combines column-weighted encodings, UMAP projection, and HDBSCAN clustering, followed by conservative consensus refinement. The pipeline was benchmarked and validated using datasets with known composition, including high-fidelity PacBio sequences. The results show that Nanopore barcoding, when paired with appropriate analysis, can recover biologically meaningful variation even in technically complex samples. The pipeline is particularly suited for specimens where divergent templates are co-amplified, including mitochondrial pseudogenes or multicopy nuclear regions like ITS. As such, it provides a generalizable framework for high-resolution Nanopore analysis of complex amplicon mixtures.

Dispersal transiently modifies the temperature dependence of ecosystem productivity after an extreme thermal fluctuation

Stark, K. — 2025-12-30

Effects of warming on ecosystem productivity are typically summarized over broad time scales, yet they emerge from communities that can reorganize in a matter of days. Temperature accelerates ecosystem productivity through predictable effects on metabolic rates, but dispersal across thermally heterogeneous metacommunities may modify this effect by redistributing communities and their underlying thermal phenotypes. Using multi-trophic freshwater mesocosm communities in which warming and dispersal were manipulated, we tested the hypothesis that increasing dispersal modifies the thermal sensitivity of gross primary productivity (GPP) through the redistribution of phytoplankton biomass, size spectra, and thermal phenotype composition along spatial thermal gradients. High dispersal temporarily weakened the thermal sensitivity of GPP after an unplanned heatwave. This effect was caused by reduced mass-specific GPP in the warmest mesocosms accompanied by regional homogenization of phytoplankton communities and the spread of poorly adapted thermal phenotypes under the highest dispersal treatments. Except immediately post-heatwave, the thermal sensitivity of GPP was robust to dispersal, despite evidence of metacommunity dynamics. These findings suggest that the temperature dependence of ecosystem metabolism can be briefly modified by dispersal after a perturbation, but remains robust under steady-state conditions.

Exercise Leverages a Post-Stress Therapeutic Window in Hypothalamic CRH Neurons to Enable Recovery from Threat Sensitization

Rojas-Carvajal, M. — 2026-01-08

Exercise provides a range of benefits for physical and mental health. It relieves stress, yet paradoxically, it recruits the bodys stress response system. Here we show that activation of corticotropin-releasing hormone cells of the paraventricular nucleus of the hypothalamus (CRHPVN) and the endocrine arm of the stress axis is necessary, but not sufficient for the beneficial effects of exercise. Specifically, glucocorticoids act synergistically with brain-derived neurotrophic factor (BDNF) on CRHPVN cells during exercise to reverse behavioral and synaptic sensitization after stress. In the absence of exercise, optogenetic activation of the tropomyosin-related kinase B (TrkB) receptor after stress is sufficient to reverse behavioral sensitization and synaptic metaplasticity. Our findings reveal a novel, time-sensitive mechanism by which exercise alleviates the impact of acute stress.

Systematic Infectome-Phenome Profiling Reveals Cryptococcal Infection-Associated Proteins Driving Immune System Remodeling and Immunization Potential

Ball, B. — 2026-01-31

Mortality and morbidity associated with invasive fungal infections continue to rise, driven by the emergence of new pathogens, increasing antifungal resistance, and expanding immunocompromised populations. Despite this growing threat, fungal biology lacks scalable, unbiased strategies to link infection-responsive fungal proteins to functional outcomes that drive virulence and immune modulation. Here, we present an integrated infectome-phenome discovery platform that combines high resolution mass spectrometry-based proteomics with systematic phenotypic profiling to globally interrogate Cryptococcus neoformans-macrophage interactions. This approach reveals coordinated host immune suppression alongside complementary fungal virulence programs and enables the unbiased prioritization of infection-associated fungal proteins. Systematic phenome fingerprinting of candidate mutant strains resolves two functional classes of putative therapeutic relevance: antifungal and antivirulence, with in vitro characterizations corroborated with a murine model of cryptococcosis. Prioritization of a conserved, previously uncharacterized virulence-associated protein, CipC, uncovers altered extracellular vesicle composition and enhanced antigenic properties. Immunization with CipC-derived vesicles elicits a robust and diversified host immune response, implicating fungal extracellular vesicles in immune remodeling and host priming. Together, these findings establish a broadly applicable framework for systematically identifying and functionally characterizing fungal drivers of infection for therapeutic and immunological target discovery with relevance across diverse human fungal pathogens.

Multi-compartment spatiotemporal metabolic modeling of the chicken gut guides dietary intervention design

Utkina, I. — 2026-02-10

Understanding the interactions between diet and the gut microbiome is critical for identifying dietary interventions that support gut health. This is of particular importance for poultry where the elimination of antibiotic growth promoters has resulted in an alarming rise in enteric infections with significant economic consequences. While the application of computational models capable of dissecting the metabolic interactions supporting gut communities has shown promise, they remain limited, largely ignoring the physiological and geographical considerations of the poultry gastrointestinal tract. To address these limitations, we developed the first multi-compartment, spatiotemporally-resolved metabolic model of the chicken gastrointestinal tract. Our six-compartment framework integrates avian-specific physiological features including bidirectional flow, feeding-fasting cycles, and compartment-specific environmental parameters. The model captured distinct metabolic specialization along the gut, with upper compartments enriched for biosynthetic pathways and lower compartments specialized for fermentation. In silico screening of 34 dietary supplements revealed context-dependent metabolic responses and predicted cellulose, starch, and L-threonine as robust enhancers of short-chain fatty acid production. A controlled feeding trial validated key predictions, particularly for butyrate, with prediction accuracy further improved through integration of trial-specific microbial community data. Our findings demonstrate that community composition is a major driver of metabolic outcomes and underscore the need for context-specific modeling. Our framework provides a mechanistic platform for rational dietary intervention design and is broadly adaptable to other animal or human gastrointestinal systems.

Diverse microbial metal resistance and novel metal cycling organisms in copper/nickel mine tailings

Chen, M. — 2026-02-27

Mine tailings contribute to environmental heavy metal contamination through the formation of acid mine drainage (AMD). Microbially-mediated processes such as iron and sulfur redox cycling influence metal mobility. Here, we applied an integrated metagenomic and metaproteomic approach to profile microbial communities across vertical geochemical gradients in legacy copper/nickel tailings in Sudbury, Ontario, Canada. From 43 samples, we recovered 454 non-redundant metagenome-assembled genomes (MAGs), revealing diverse populations within the Actinobacteriota, Desulfobacterota, and uncultured lineages such as Candidatus Eremiobacterota and SZUA-79. Functional profiling identified 301 putative iron- and sulfur-cycling MAGs, including those within the Ca. Eremiobacterota and SZUA-79 phyla. Metal resistance genes were widespread and diverse, with abundances that did not correlate with measured metal concentrations. Proteomic data confirmed in situ expression of selected metal resistance genes and iron/sulfur metabolism genes, despite limited protein recovery from this challenging matrix. Our findings highlight both the depth of microbial diversity in metal resistance and metal biogeochemical cycling in mining waste, as well as the technical challenges that currently limit genomic and proteomic sequencing coverage in low-biomass, metal-rich matrices. This work also presents new protocols for multi-omics data capture and analysis from metal contaminated environments, including new protein extraction and bioinformatic gene annotation tools. HighlightsO_LIMetagenomics recovered 454 non-redundant MAGs from copper/nickel mine tailings, revealing high taxonomic novelty including uncultured Ca. Eremiobacterota and SZUA-79 lineages. C_LIO_LIA custom HMM-based pipeline targeting metal resistance genes uncovered a widespread and diverse set of metal resistance genes across MAGs in tailings, which did not correlate with metal concentrations. C_LIO_LIMetaproteomics validated in situ expression of both iron/sulfur cycling genes and metal resistance proteins, although low biomass and contamination limited proteomic sequencing coverage. C_LI

Biological implications of a detailed repeat annotation in Octopus vulgaris

Bonar, M. — 2026-03-05

Octopuses are phenotypically distinctive organisms, and recent genomic work raises questions about the contributions of transposable elements (TE) to their genomic architecture. We leveraged a robust repeat annotation pipeline, in combination with manual and automated curatorial techniques, to produce a more comprehensive repeat annotation of Octopus vulgaris. This revealed that [~]66% of the genome are repeats, in contrast to previous estimates of 43-50%. Whereas previous studies of TE expansion in Octopus bimaculoides identified two bursts of activity, 25 and 56 MYA, our re-annotation revealed four such expansions at 18, 25, 33, and 56 MYA. We further identified a landscape of TE hot- and cold spots. This much refined TE timescape and landscape will serve as a useful basis for understanding TE contributions to O. vulgaris evolution, and also for identifying factors contributing to variation in the TE community across genomic space and evolutionary time.

Dissecting genetic variance structure and evaluating genomic prediction models for single-cross hybrids derived from Stiff Stalk and Non-Stiff Stalk maize heterotic groups

Godoy, J. C. — 2026-03-13

The early 20th-century discovery of heterosis and the establishment of heterotic groups transformed maize (Zea mays L.) into a keystone of global agriculture. However, maize breeding faces two significant challenges: the gradual decline of general combining ability (GCA) variance within heterotic groups and the impracticality of testing all possible single crosses in the early stages of a breeding program. Here, we developed genomic best linear unbiased prediction (GBLUP)-based multi-kernel models, using additive and two alternative non-additive genomic relationship matrices, to estimate the variance components associated with the GCA of Stiff Stalk (SS) and Non-Stiff Stalk (NSS) heterotic groups and the specific combining ability (SCA) arising from their crosses. We further applied these models to predict the performance of untested single-cross combinations under varying levels of parental information. We showed that the SS and NSS groups retained significant GCA variance across traits in both early- and late-maturity groups. The SS group, in contrast, exhibited no detectable GCA variance in grain yield for the intermediate-flowering subset of hybrids, highlighting a limitation for future genetic improvement. Furthermore, our results showed that GBLUP-based multi-kernel models effectively identified superior hybrids when parental information was available. In the absence of this information, however, these models underperformed compared to covariance-based approaches. Both types of non-additive matrices produced similar results, affirming the robustness of the inferred genetic architecture. Overall, this study sheds light on the future use of US maize commercial germplasm and demonstrates how GBLUP-based multi-kernel models can improve the efficiency of hybrid breeding programs.

Shifting forage selection subsidizes seasonal resource scarcity

Hendrix, J. G. — 2026-03-17

O_LINitrogen (N) is limiting for terrestrial herbivores, particularly over winter. Caribou (Rangifer tarandus) have adapted to seasonal scarcity of N by accruing muscle mass during the growing season when N is more abundant. C_LIO_LINitrogen stored in muscle tissue is then relied upon during winter to compensate for dietary deficits. Once their diet shifts from N-rich vascular plants to N-poor lichen over winter, caribou can lose [~]30% of their muscle mass. As catabolized N is shed in urine on wintering grounds, caribou could act as elemental transport across seasons and landscapes. Furthermore, if deposited N is taken up by lichen or other winter forage, it might enrich the nitrogen-poor winter diet of caribou in the future. C_LIO_LIWe tested this potential transport via three steps. We analysed Cladonia spp. lichen and vascular plants upon which caribou forage across Fogo Island, Newfoundland, using %N content as our metric of forage quality. We then compared seasonal habitat selection responses to forage quality by caribou using integrated step selection analyses. In summer, caribou selected areas with higher vascular plant %N but did not select nor avoid Cladonia quality. In contrast, caribou selected sites with higher quality Cladonia in winter but responded neutrally to vascular plant quality. C_LIO_LIWe compared seasonal distributions of caribou to determine whether nitrogen consumed in summer and deposited in winter would occur in spatially discrete locations. Population-level kernel density estimates for summer and winter in this island herd were mostly non-overlapping, lending credence to the potential landscape effects of N transport. C_LIO_LIWhen viewed together with established seasonal changes in woodland caribou physiology, sociality, and forage preferences, the shifts in habitat selection and seasonal ranges we observe here could serve as an adaptive strategy for caribou to recycle N and mitigate winter nutrient scarcity. C_LI

A robust negative association between estimated tumour circadian clock function and survival in early stage breast cancer

Vasilyev, V. — 2026-03-18

Recent studies have established that the circadian clock influences onset, progression and therapeutic outcomes in a number of chronic conditions including cardio-metabolic diseases and cancer. For the latter, they also suggest that chronotherapy offers the potential to refine current treatments and improve the development of future anti-cancer medicines. Therefore, there is a need for tools to measure the functional state of the tumoural circadian clock in patients. We have previously developed a model-led machine-learning algorithm called TimeTeller which has the potential to provide such a tool. Here we demonstrate its potential for clinical relevance by a study of 1286 breast cancer patients in which we characterise the nature of the circadian clock disruption in their tumours and demonstrate a strong nonlinear association between 10-year survival and TimeTellers tumoural clock disfunction score {Theta}. We find that good tumour clock function is antagonistic to survival.

Pareto fronts reveal constraints on the evolution of niche-determining traits in phytoplankton

Laurich, J. R. — 2026-03-23

Trade-offs are central to biodiversity because they prevent the emergence of dominant phenotypes by limiting the simultaneous optimization of multiple fitness components. Yet trade-offs are often difficult to detect empirically when variation in overall performance produces positive correlations that mask underlying constraints. Here we use Pareto fronts--boundaries that capture optimal trade-off solutions--to test for evolutionary constraints on niche-determining traits in phytoplankton, including minimum nutrient requirements, thermal breadth, salt tolerance, and population growth rates. Using experimentally evolved Chlamydomonas reinhardtii populations subjected to nutrient and salt stress, we detected widespread Pareto fronts limiting the joint optimization of growth rate and niche-determining traits, thereby restricting the emergence of multivariate stress tolerance. Importantly, Pareto fronts revealed trade-offs even when underlying trait correlations were positive. We found that the structure of trait covariation behind Pareto fronts strongly predicted evolutionary outcomes: populations moved toward Pareto-optimal phenotypes primarily when trait correlations were neutral or positive, whereas negative trait correlations were associated with limited evolutionary optimization. Extending this framework across phytoplankton diversity, we compiled niche-determining traits for 299 phytoplankton taxa. At a macroevolutionary scale, we detected significant Pareto fronts constraining the evolution of niche-determining traits in phytoplankton. These fronts, however, did not always recapitulate the structure of trade-offs evident among C. reinhardtii populations, suggesting that forces that dictate microevolutionary outcomes, such as genetic correlations, can be resolved across macroevolutionary time. Together, our results highlight that evolutionary trajectories may differ across scales, but that fundamental limits on multivariate trait optimization persist across phytoplankton. Significance StatementTrade-offs among biological traits are central to evolutionary theory but often prove difficult to detect empirically. Here, we apply Pareto fronts--a framework borrowed from economics and engineering--to detect and reveal trade-offs among key niche-determining traits in phytoplankton. By combining experimental evolution in the laboratory with a synthesis of ecological traits across 299 taxa, we demonstrate widespread limits on the simultaneous optimization of growth rate, nutrient competition, salt tolerance, and thermal breadth. Importantly, Pareto fronts reveal trade-offs even when conventional correlation-based approaches fail, uncovering evolutionary constraints that remain hidden in trait correlations. These results show that trade-offs shape phenotypic variation across both micro- and macroevolutionary scales and impose fundamental limits on phytoplankton responses to multiple environmental stressors.

Temperature-dependent performance scales with maximum heat tolerance across ectotherms

Cicchino, A. S. — 2026-03-24

Projecting species responses to changing temperatures remains a major challenge in ecology. Central to this effort is harnessing our understanding of species thermal physiological traits, which underlie ectotherm fitness. These traits are typically characterized by describing performance across temperatures (thermal performance curve, TPC), and/or tolerance limits, which capture endpoints of biological failure. Despite their importance, we still lack an understanding of the functional relationship between these traits, limiting our ability to integrate them into comprehensive vulnerability assessments. Using a synthesized dataset of >100 ectotherms, we tested how heat tolerance (CTmax) relates to key TPC traits: thermal optima, thermal maxima, and the supra-optimal range of temperatures where performance is positive. Across taxa, TPC traits were positively related to CTmax, demonstrating a link between heat tolerance and temperature-dependent performance at sub-critical temperatures. While acute locomotor performance scaled proportionally with CTmax, metabolic processes and sustained locomotion scaled sub-proportionally, suggesting decoupling of CTmax and performance among high-CTmax species. This suggests that using CTmax as a comparative metric may overestimate thermal safety margins for metabolic processes critical to growth. Our results indicate that while CTmax and TPCs reflect shared underlying constraints--particularly in acute neuro-muscular traits--their relationship is dependent on timescale and the TPC response trait. Our findings connect our understanding of the processes that maintain performance over thermal gradients with those that cause performance to fail, improving our ability to project species persistence in a warming world. SignificanceClimate warming is increasingly reshaping the thermal environments that govern species persistence worldwide. Predicting vulnerability requires integrating multiple aspects of thermal biology, yet relationships among widely used thermal traits remain poorly understood. By synthesizing data from more than 100 ectotherm species, we quantify links between acute heat tolerance and traits describing sustained biological function across temperatures. We show that performance at relatively benign temperatures and performance at thermal extremes are coupled, but this coupling is strongly process and timescale dependent, with close correspondence for short term locomotion but weaker coupling for metabolic processes. Our results link the processes that maintain performance across temperatures with those that cause failure, fundamentally advancing our projections of species performance in a warming world.

A fungal effector targets the chloroplast to support biotrophy by balancing disease and plant health

Damoo, D. — 2026-03-26

Fungal pathogens are responsible for substantial crop losses worldwide. There is a pressing need to develop crops with improved disease resistance, especially given that climate change and human activities are exacerbating crop diseases. Our understanding of the molecular mechanisms by which fungi cause disease is incomplete. To address this limitation, we employed proteomics to identify candidate effector proteins from the pathogenic fungus Ustilago maydis that co-purified with the chloroplasts of maize host plants during infection. We specifically characterized the role of one putative chloroplast-associated effector, UmPce3, using heterologous expression in the non-host plant Arabidopsis thaliana. We discovered that UmPce3 interacts with the chloroplast DEAD-box RNA helicase, AtRH3. Phenotypes associated with the expression of UmPce3 in Arabidopsis mirrored those of plants with impaired AtRH3 function and included interference with chloroplast assembly, an impact on photosynthesis, and altered resistance to biotic and abiotic stresses. Support for RH3 as a bona fide effector target was obtained by identifying parallel phenotypic influences of UmPce3 in maize and by demonstrating an interaction between UmPce3 and maize ZmRH3b, an ortholog of AtRh3. Notably, UmPce3 contributes to biotrophy by promoting the virulence of U. maydis on maize seedlings and dampening virulence in plants challenged with salinity as an abiotic stress. Overall, this work highlights the chloroplast as a target of fungal pathogenesis and identifies RH3 as a potential hub for pathogen manipulation of organelle function to balance fungal proliferation and host health in support of biotrophy. Short summaryThe chloroplast plays a key role in plant immunity, in addition to its central contributions to photosynthesis, metabolism, and tolerance of abiotic stresses. The effector UmPce3 of the maize pathogen Ustilago maydis targets the DEAD-box RNA helicase RH3 in host plants to manipulate chloroplast function and enhance fungal pathogenesis. Unexpectedly, UmPce3 also influences host tolerance to salt stress thereby balancing the plant response to biotic and abiotic stressors in support of biotrophic development.

Chromatin remodelling enables enhancer resetting to facilitate the ERK transcriptional response

Ragheb, R. — 2026-03-28

During development, cellular identity is ultimately determined by transcriptional output: lineage-specific genes must be activated, while genes associated with alternative fates must be repressed. This process depends on the activity of chromatin remodelling complexes, which regulate the accessibility of transcription factors to chromatin regulatory elements. In addition, cellular identity is shaped by exposure to intercellular signals. Understanding the mechanisms by which extracellular signals are translated into changes in the transcriptional program is essential for understanding cell fate decisions during development, as well as in disease conditions such as cancer. Here we describe a rapid and widespread enhancer resetting event in response to ERK signalling in mouse ES cells. This process occurs in two distinct phases: an immediate, genome-wide alteration in transcription factor binding dynamics at regulatory regions which is dependent on the release of paused RNA Polymerase II, followed by the re-establishment of a context appropriate, stable chromatin state. We demonstrate that the chromatin remodelling complex NuRD is required for this reestablishment phase and for the appropriate transcriptional response to ERK signalling. We propose that enhancer resetting places genomic regulatory regions in a state which is permissive to the exchange of transcription factors in order to establish a new, stable enhancer topology enabling rapid yet precise transcriptional response to extracellular signals.

Estimation of chloroplast macromolecular complex copy numbers and subunit stoichiometries during the Chlamydomonas reinhardtii cell cycle

Schmollinger, S. — 2026-04-01

An unbiased, quantitative view of biomolecules in a living cell is a prerequisite for accurate modeling approaches and informs our understanding of cellular metabolism at scale. In this work, we used the total protein approach (TPA), in which the total protein mass of a given proteomics sample is used as a calibrator for absolute protein quantification, to determine protein abundances during the Chlamydomonas reinhardtii diurnal cycle. We use external, independently measured quantitative markers (metals, pigments) to assess the absolute protein abundances in unlabeled whole cell extracts. We calculate protein abundances in fg / cell of 7322 Chlamydomonas proteins, 2266 of which were captured in every time point, including the major proteins involved in the light reactions, photoprotection, proteostasis and fatty acid metabolism during a cell cycle. As expected, Rubisco large and small subunits are present in a 1:1 stoichiometry, with the large subunit being the most abundant protein in our data set, averaging 5.05 x 106 molecules per cell, reflecting 2.7% of the total protein mass. We noticed that PSII is the most abundant complex involved in the light reactions with 2.08 x 106 complexes per cell. PSI averages 1.75 x 106 complexes per cell and cytochrome b6f averages 0.77 x 106 complexes per cell. The TPA is a robust tool to study proteome dynamics quantitatively, while avoiding artefacts due to biochemical fractionation. Our proteome data set with an unprecedented temporal resolution is a valuable resource to assess protein abundances during the cell cycle in the reference alga Chlamydomonas.

Disentangling shape and size in a population of unusually large Threespine Stickleback (Gasterosteus aculeatus) from Vancouver Island, British Columbia

Perry, S. — 2026-04-03

Sarita Lake, British Columbia houses a distinctive population of threespine stickleback (Gastrosteus aculeatus L.) with a phenotype characterized by unusually large individuals relative to nearby conspecifics. We tested the hypothesis that members of this population are not isometrically larger but rather exhibit variation in allometric trajectories that reflect changes in developmental timing impacting the developmental-genetic architecture of the phenotype. We used 3D geometric morphometrics to characterize the size and shape of skulls, pectoral girdles and pelvic girdles from a sample of individuals from nearby freshwater and marine populations and compare them to a sample from Sarita Lake. We showed that individuals from the Sarita Lake population are larger in each body region compared to most other populations examined. Further, these individuals have dorsally expanded skulls and relatively robust pelvic armour. We also showed that the relationship between size and shape is differently structured among body regions and is heavily influenced by non-uniform sexually-mediated variation across populations sampled. Our results reflect complex underlying developmental trajectories, and we suggest that the large phenotype observed may be driven by fecundity selection on female size in combination with a limnetic trophic niche and relatively increased predation pressure in Sarita Lake.

ZDHHC13 is a likely pseudoenzyme protein S-acyltransferase that functions via a non-canonical mechanism

Petropavlovskiy, A. A. — 2026-04-22

S-acylation is the addition of fatty acids to cysteine residues to regulate protein function and localization. S-acylation is catalyzed by the ZDHHC (Asp-His-His-Cys) family of protein S-acyltransferases (PATs), which S-acylate protein substrates by first auto-S-acylating the catalytic cysteine of the DHHC active site followed by transfer to the substrate. ZDHHC13 and ZDHHC17 are related ankyrin repeat domain (ANK) PATs that S-acylate multiple neuronal proteins, including huntingtin (HTT), the protein mutated in Huntington disease. However, unlike ZDHHC17 and other human PATs, ZDHHC13 possesses a non-canonical DQHC active site. As the first histidine is essential for auto-S-acylation, it is unclear if ZDHHC13 is catalytically active. Our phylogenetic analysis of eukaryotic ANK-containing PATs shows that ZDHHC13 orthologues are more divergent compared to ZDHHC17. While the ZDHHC17 DHHC is highly conserved, the motif varies among ZDHHC13 orthologues, with some vertebrate lineages containing a serine in place of the catalytic cysteine. Interestingly, we found that the ZDHHC13 S-acylation is lower than that of ZDHHC17, but the ZDHHC13 catalytic cysteine is indeed S-acylated. While expression of wild type (WT) ZDHHC13 in ZDHHC13 deficient HEK293T cells increased S-acylation of a HTT1-588 fragment, surprisingly, expression of catalytically dead DQHS ZDHHC13 was still able to facilitate HTT1-588 S-acylation equally. This suggests the ZDHHC13 catalytic cysteine is not required for S-acylation of target proteins, suggesting ZDHHC13 may coordinate another PAT. Indeed, we identified ZDHHC13 in high-molecular weight complexes. Our results indicate that ZDHHC13 is a likely pseudoenzyme that may function via a non-conventional mechanism reliant on other PATs. This work broadens our understanding of the function of this non-canonical PAT.

SbmA coordinates iron homeostasis and antimicrobial susceptibility in Klebsiella pneumoniae through phosphorylation-dependent regulation

Reitzel, C. — 2026-04-27

Iron is an essential nutrient that underpins fundamental biological processes, yet its bioavailability is severely restricted during infection due to oxidation and host-mediated sequestration. In Gram-negative pathogens, such as Klebsiella pneumoniae, iron limitation imposes a critical selective pressure, necessitating tightly regulated acquisition systems to support growth, virulence, and survival. While canonical pathways for iron uptake are well characterized, regulatory mechanisms coordinating these processes remain incompletely understood. Here, we applied mass spectrometry-based phosphoproteomics to identify iron-responsive regulatory events associated with bacterial iron homeostasis. This approach revealed iron-dependent phosphorylation of SbmA, a conserved inner membrane transporter previously implicated in the uptake of antimicrobial peptides and related substrates. Functional characterization demonstrated that deletion of sbmA results in reduced intracellular iron levels and altered cellular morphology, supporting a role in iron acquisition. Complementary proteome mapping of {Delta}sbmA revealed compensatory production of siderophore receptors and TonB-dependent transport systems, further implicating SbmA in maintaining iron balance. Leveraging these findings, integration with high-throughput drug screening identified a compound that exploits SbmA-mediated transport to inhibit bacterial growth, highlighting its potential as a therapeutic entry point. Collectively, this work uncovers a previously unrecognized role for SbmA in iron homeostasis and demonstrates the power of phosphoproteomics to identify condition-specific regulators of essential bacterial pathways. These findings position SbmA as a promising target for antimicrobial development in K. pneumoniae.

Iron-responsive phosphorylation of TolQ modulates cell envelope integrity and antibiotic susceptibility in Klebsiella pneumoniae

Reitzel, C. — 2026-04-27

Klebsiella pneumoniae is an opportunistic bacterial pathogen associated with high morbidity and mortality, exacerbated by the rapid emergence of resistance to last-resort antibiotics, such as carbapenems. Adaptation to nutrient limitation, particularly fluctuations in metal availability, is critical for bacterial survival and virulence, yet the regulatory mechanisms coordinating these responses remain incompletely understood. Protein phosphorylation represents a key post-translational modification governing bacterial physiology and offers a promising avenue for identifying novel antimicrobial targets. Here, we applied mass spectrometry-based phosphoproteomics to define nutrient-responsive signaling networks in K. pneumoniae under varying iron and zinc conditions. This analysis identified iron-dependent phosphorylation of TolQ, a conserved inner membrane component of the Tol-Pal system that maintains cell envelope integrity. Structural modeling predicted that phosphorylation modulates TolQ-TolR conformation, suggesting a mechanism by which iron availability regulates Tol-Pal function. Functional characterization demonstrated that deletion of tolQ results in reduced bacterial viability, increased susceptibility to host immune clearance, and heightened sensitivity to antibiotic treatment. To further explore the therapeutic potential of this pathway, we integrated high-throughput compound screening with computational modeling and identified small molecules that phenocopy {Delta}tolQ. Collectively, these findings reveal a previously unrecognized link between iron availability and phosphoregulation of the Tol-Pal system and establish TolQ as a critical mediator of bacterial survival. This work highlights phosphoproteomics as a powerful strategy to uncover regulatory vulnerabilities and identify targets for antimicrobial development in drug-resistant pathogens.

Thinking outside of the box: Refining rat housing to improve welfare

O'Malley, C. — 2026-05-04

Standard rat housing may impede species-typical behaviors and impact rat welfare and research outcomes. This research investigated the effects of housing on behavioral and physiological outcomes of rats through the use of modified large animal cages for housing, and was conducted in two studies. Study A: 70 Sprague Dawley (SD) rats (34 males, 36 females; 5 wk old) were randomly assigned to standard polycarbonate shoebox cages (C: 733.9cm2) or modified stainless steel primate cages (T: 10,416cm2) for 18 days. In Study B: 48 SD rats (24 males, 24 females; 7.5 wk old) were held in T housing for 90 days to assess long term impacts. All rats received gentle handling for 15s 3x/week. Rats were assessed for body weight, anxiety-like behavior in an elevated plus maze, response during a voluntary human approach test, and overall home cage behavior, posture, and space usage. Data were analyzed using generalized linear mixed models, with sex and treatment as fixed effects, and cage as the random effect. The results of study A suggest that the modified large animal cages (T) had positive impacts on rat behavior and welfare. T rats were less anxious (P=0.038) and more active (P<0.0001) and explorative (P=0.0003) compared to C rats. In both groups, activity levels declined towards the end of the 18-day study period (P<0.0001). For study B, similar patterns were observed, with rats becoming more inactive (P<0.0001) over 90 days. However, rats spent significant time on elevated shelves in T housing, which increased throughout the study (P<0.0001), suggesting continued use of the resources the housing provided. In both studies, there were no differences in latency to approach humans (P>0.05), but T rats spent less time in contact with human handlers, suggesting differences in motivation to interact with humans that should be explored further.

Ethylene-Gibberellin Crosstalk Drives Phenotypic Sex Changes in Cannabis sativa

Roy, J. — 2026-05-14

Abstract/SummarySex expression in Cannabis sativa is determined by XX/XY sex chromosomes but remains plastic, with ethylene inhibition inducing male flowers on XX plants and ethylene release inducing female flowers on XY plants. Although ethylene is a central regulator of this process, the contribution of gibberellin signaling to cannabis sex reversal remains poorly defined. Here, we reconstructed the GA biosynthesis, regulation, and signaling pathway in C. sativa and profiled GA-related gene expression during chemically induced sex reversal. Orthology-based searches identified 50 putative C. sativa GA-related genes, widely distributed across the genome, with the X chromosome harboring 11 genes, including six within the non-recombining region. Transcriptomic analyses across vegetative baseline, early post-treatment leaves, and developing flowers showed that expression profiles were broadly similar between XX and XY plants at day 0, weakly perturbed at day 1, and strongly structured by floral phenotype at day 14. Early responses were limited to downregulation of CsGA3ox1 in ethephon-treated XY plants and CsGASA1 in STS-treated XX plants. By day 14, sex reversal was associated with differential expression of key genes, including CsGA1, multiple GA20ox orthologs, CsGID1B, CsSLY2, and several GASA genes, indicating broad remodeling of GA regulation. Our findings position the GA pathway as a downstream module of ethylene-driven sex reversal in C. sativa, with GA activity tracking floral sexual identity, extending the framework of sexual plasticity beyond ethylene, and identifying candidate genes for functional validation and the development of sex-stable cultivars.

Structural Characterization of Calcium-Dependent Calmodulin-Calmidazolium Binding using Capillary Vibrating Sharp-Edge Spray-based Native Mass Spectrometry and In-Droplet Hydrogen Deuterium Exchange Mass Spectrometry

Courtney, K. C. — 2026-05-19

Native mass spectrometry (nMS) is a powerful tool for analyzing biomolecules and their complexes under near native conditions. The preservation of the native state depends strongly on the ionization methods used to transfer intact molecules from solution to gas phase. In this work, capillary vibrating sharp-edge spray ionization (cVSSI)- based nMS and in-droplet hydrogen deuterium exchange mass spectrometry (HDX-MS) were used to evaluate calcium-dependent interactions between calmodulin and calmidazolium (CDZ). We found that cVSSI produced a narrow charge-state-distribution (CSD) with low average charge states indicating that this method preserved the native-like state. cVSSI was also able to resolve stepwise Ca2+-binding containing one to four Ca2+-bound species of the protein. In absence of Ca2+, no detectable CDZ-binding was observed. However, CDZ-binding was observed when calmodulin was fully loaded with Ca2+. CDZ-binding to the protein caused marked redistribution of the CSD toward lower charge states, consistent with ligand-induced stabilization of the protein into a more compact conformation. The apparent dissociation constant (Kd) of the interaction was determined to be 261 {+/-} 29 nM and 126 {+/-} 17 nM from Langmuir and quadratic binding models, respectively. Complementary in-droplet HDX-MS showed an approximately 23% reduction in deuterium uptake upon ligand binding indicating reduced solvent accessibility and increased structural stabilization supporting nMS findings. Together, these results demonstrate that cVSSI-based nMS coupled with in-droplet HDX-MS provides an integrated platform for simultaneously resolving metal loading, ligand binding, binding affinity, and ligand-induced conformational changes. This approach complements traditional structural methods by enabling direct interrogation of dynamic, metal-dependent protein-ligand interactions in their native states.

Activity Patterns Structure Food Web Interactions Through Time

Scott, A. M. — 2026-05-22

Many mobile animals move to locate and consume resources, making energy gain and growth dependent on activity. Yet the role of activity in shaping predator-prey interactions in food webs has not been broadly considered. Here, we synthesize empirical examples to examine how three activity traits (mean, variance, and timing) vary among taxa (fish, mammals, birds) and between predators and prey across temporal scales. We then use predator-prey models to explore how these diverse activity patterns influence stability. Motivated by emerging activity patterns, our theory shows that fluctuating activity rates can drive predator-prey interaction strengths with major consequences for stability. Future research is needed on activity trait patterning, links between activity and attack rates, and the consequences of activity for predator-prey interactions to whole food webs. This is especially critical as human-driven changes to abiotic cues increasingly alter animal activity rates and may rewire food webs.

A pistillate-biased epidermal regulatory program underlies glandular trichome development in Cannabis sativa

Monthony, A. S. — 2026-05-29

Abstract/SummaryGlandular trichomes are specialized epidermal structures on Cannabis sativa L. inflorescences that synthesize and store cannabinoids and terpenoids, making them central to the species economic and medicinal value. Although trichome development is a multistage genetic process, its regulatory basis in C. sativa remains poorly understood, particularly with respect to sexual dimorphism and sex plasticity. Here, we combined orthology-based gene discovery with transcriptomic profiling to investigate trichome development across four floral phenotypes: female flowers (FF), male flowers (MF), induced male flowers on XX plants (IMF), and induced female flowers on XY plants (IFF). Using trichome development-related genes from Arabidopsis thaliana, a model for unicellular non-glandular trichome development, and Solanum lycopersicum, a model for multicellular glandular trichomes, we identified and mapped 53 candidate C. sativa trichome development regulator genes (CsTDRGs). The CsTDRG set did not support a simple Arabidopsis- or tomato-like model, but instead included Arabidopsis-like epidermal fate components, including MBW-related regulators and GL2, alongside tomato-like multicellular trichome regulators, including MIXTA-like, HD-ZIP IV, WOX, MTR, GRAS, and hormone-associated candidates. RNA-seq analysis showed that CsTDRG expression was more strongly associated with floral phenotype than chromosomal sex. Genes with preferential expression in pistillate tissues were CsTT8, CsMYC1/GL3, CsGL2, CsYABBY4 and CsMIXTA-like1/MYB106, whereas CsMTR1, CsGRAS9, and CsCKX3 were upregulated in male (MF and IMF) flower. These findings suggest that sexually dimorphic trichome development in C. sativa reflects differential regulation of a shared developmental toolkit that combines conserved epidermal fate components with multicellular and glandular trichome regulatory modules.