Comparative Analysis of T Cell Spatial Proteomics and the Influence of HIV Expression As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new, unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses we found that classifier performance in this system was organelle-dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine (SVM) learning for mitochondrial and ER proteins, but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, BANDLE, on SVM-classified data. Comparative BANDLE analysis of cells induced to express the wild-type viral genome vs. cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as TCR signaling components and coatomer complex. Lastly, we found that SVM classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.
Visualizing functional human profilin in cells and in vitro applications Profilin is an essential regulator of actin and microtubule dynamics and therefore a critical control point for the normal division, motility, and morphology of cells. Most studies of profilin have focused on biochemical investigations using purified protein because high cellular concentrations present challenges for conventional imaging modalities. In addition, past studies that employed direct labeling or conventional fusion protein strategies compromised different facets of profilin function. We engineered two versions of tagged profilin that retain native activities with respect to phosphoinositide lipids, actin monomers, formin-mediated actin assembly, and microtubule polymerization. mApple-profilin-1 directly binds to dimers of tubulin (kD = 1.7 {micro}M) and the microtubule lattice (kD = 10 {micro}M) to stimulate microtubule assembly. In cells, Halo-tagged profilin-1 fully rescues profilin-1(-/-) cells from knockout-induced perturbations to cell shape, actin filament architecture, and microtubule arrays. In cells expressing Halo-profilin, we visualized a subset of individual molecules of profilin-1 by titrating fluorescent Halo-ligands. Further, we combined this imaging approach with specific function-disrupting point-mutants in profilin to visualize dynamic profilin associated with microtubules in live-cells. Thus, these tagged profilins are reliable tools for studying the dynamic interactions of profilin with actin or microtubules in live-cell or in vitro applications.
Heterogeneous recruitment abilities to RNA polymerases generate nonlinear scaling of gene expression level with cell volume While most genes expression levels are proportional to cell volumes, some genes exhibit nonlinear scaling between their expression levels and cell volume. Therefore, their mRNA and protein concentrations change as the cell volume increases, which often have crucial biological functions such as cell-cycle regulation. However, the biophysical mechanism underlying the nonlinear scaling between gene expression and cell volume is still unclear. In this work, we show that the nonlinear scaling is a direct consequence of the heterogeneous recruitment abilities of promoters to RNA polymerases based on a gene expression model at the whole-cell level. Those genes with weaker (stronger) recruitment abilities than the average ability spontaneously exhibit superlinear (sublinear) scaling with cell volume. Analysis of the promoter sequences and the nonlinear scaling of Saccharomyces cerevisiaes mRNA levels shows that motifs associated with transcription regulation are indeed enriched in genes exhibiting nonlinear scaling, in concert with our model. Sablefish (Anoplopoma fimbria Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions. MTORC2 is a physiological hydrophobic motif kinase of S6 Kinase 1. Ribosomal protein S6 kinase 1 (S6K1), a major downstream effector molecule of mTORC1, regulates cell growth and proliferation by modulating protein translation and ribosome biogenesis. We have recently identified eIF4E as an intermediate in transducing signals from mTORC1 to S6K1 and further demonstrated that the role of mTORC1 is restricted to inducing eIF4E phosphorylation and interaction with S6K1.This interaction relieves S6K1 auto-inhibition and facilitates its hydrophobic motif (HM) phosphorylation and activation as a consequence. These observations underscore a possible involvement of mTORC1independent kinase in mediating HM phosphorylation. Here, we report mTORC2 as an in-vivo HM kinase of S6K1. We show that rapamycin resistant S6K1 truncation mutant {Delta}NH{Delta}CT, continues to display HM phosphorylation with selective sensitivity toward torin-1. We also show that HM phosphorylation of wildtype S6K1and {Delta}NH{Delta}CT depends on the presence of mTORC2 regulatory subunit-rictor. Furthermore, truncation mutagenesis analysis highlights the involvement of a conserved 19 amino acid stretch of S6K1 in mediating mTORC2 dependent HM phosphorylation. We finally show that deletion of this amino acid stretch leads to complete loss of HM phosphorylation regardless of the presence of functional TOS motif. Our data identifies mTORC2 as a physiological HM kinase that can activate S6K1 after its auto-inhibition is overcome by mTORC1. We, therefore, propose a novel mechanism for S6K1 regulation where mTOR complex 1 and 2 act in tandem to activate the enzyme. Comparative proteome signatures of trace samples by multiplexed Data-Independent Acquisition Single cell transcriptomics has revolutionized our understanding of basic biology and disease. Since transcript levels often do not correlate with protein expression, it is crucial to complement transcriptomics approaches with proteome analyses at single cell resolution. Despite continuous technological improvements in sensitivity, mass spectrometry-based single cell proteomics ultimately faces the challenge of reproducibly comparing the protein expression profiles of thousands of individual cells. Here, we combine two hitherto opposing analytical strategies, DIA and Tandem-Mass-Tag (TMT)-multiplexing, to generate highly reproducible, quantitative proteome signatures from ultra-low input samples. While conventional, data-dependent shotgun proteomics (DDA) of ultra-low input samples critically suffers from the accumulation of missing values with increasing sample-cohort size, data-independent acquisition (DIA) strategies do usually not take full advantage of isotope-encoded sample multiplexing. We developed a novel, identification-independent proteomics data-analysis pipeline that allows to quantitatively compare DIA-TMT proteome signatures across hundreds of samples independent of their biological origin, and to identify cell types and single protein knockouts. We validate our approach using integrative data analysis of different human cell lines and standard database searches for knockouts of defined proteins. These data establish a novel and reproducible approach to markedly expand the numbers of proteins one detects from ultra-low input samples, such as single cells. Predicting the zoonotic capacity of mammals to transmit SARS-CoV-2 Back and forth transmission of SARS-CoV-2 between humans and animals may lead to wild reservoirs of virus that can endanger efforts toward long-term control of COVID-19 in people, and protecting vulnerable animal populations that are particularly susceptible to lethal disease. Predicting high risk host species is key to targeting field surveillance and lab experiments that validate host zoonotic potential. A major bottleneck to predicting animal hosts is the small number of species with available molecular information about the structure of ACE2, a key cellular receptor required for viral cell entry. We overcome this bottleneck by combining species ecological and biological traits with 3D modeling of virus and host cell protein interactions using machine learning methods. This approach enables predictions about the zoonotic capacity of SARS-CoV-2 for over 5,000 mammals -- an order of magnitude more species than previously possible. The high accuracy predictions achieved by this approach are strongly corroborated by in vivo empirical studies. We identify numerous common mammal species whose predicted zoonotic capacity and close proximity to humans may further enhance the risk of spillover and spillback transmission of SARS-CoV-2. Our results reveal high priority areas of geographic overlap between global COVID-19 hotspots and potential new mammal hosts of SARS-CoV-2. With molecular sequence data available for only a small fraction of potential host species, predictive modeling integrating data across multiple biological scales offers a conceptual advance that may expand our predictive capacity for zoonotic viruses with similarly unknown and potentially broad host ranges. Pan-cancer HLA Gene-mediated Tumor Immunogenicity and Immune Evasion Human Leukocyte Antigen (HLA) expression contributes to the activation of anti-tumor immunity through interactions with T cell receptors. However, pan-cancer HLA-mediated immunogenicity and immunoediting mechanisms has not been systematically studied. In a retrospective analysis of 33 tumor types from the Cancer Genome Atlas, we uncovered HLA class I and class II differential expression, which outperformed traditional clinical metrics in predicting patient survival. We also characterized the distribution of HLA supertypes across cancers and showed that patients with high HLA allelic diversity and gene expression had better prognosis. Immune microenvironments with varied survival outcomes could be predicted using a neural network model trained on HLA expression data. Furthermore, we identified a subset of tumors which upregulated HLA class I but not class II genes and exploited HLA-mediated escape strategies. Our results suggest the potential of using HLA expression data to predict patient prognosis. Taken together, we emphasize the crucial role of HLA upregulation in shaping prolonged anti-tumor immunity.

SynopsisIn a retrospective analysis of 11080 patients of 33 TCGA cancer types, we showed HLA class I and class II differential expression shape various immune microenvironments and tumor immunoediting mechanisms, predicting tumor immunogenicity and patient survival. Bamboos flower after the return of almost the same sun-moon phasing as at seedling emergence All Asian woody bamboo species of economic importance are semelparous. They remain vegetative during time intervals that are specific to each species and range from three to 120 years, with notable concentrations around a series of values (3, 7-8, 14-17, 29-36, 42-48, 61-64, and 120 years). Then, they flower gregariously within a short period. Entire forests temporarily disappear during these periods, and the dates of these dramatic events have been recorded over the last 200 years.

While searching for a correlated environmental cue, I have found that the concentrations of flowering cycles were highly correlated with the series of successive returns of almost the same sun-moon phasing as at seedling emergence. On basis of knowledge on plant photoperiod sensitivity, I hypothesize that bamboo plants i) run a lunar cellular clock that is set at the full moon, ii) retain in their cellular memory the exact sun-moon phasing of the year of their emergence as seedlings, and iii) inhibit flowering until the occurrence of a unique, species-specific sun-moon phasing that is shifted by a precise amount from the sun-moon phasing at their emergence. Recent evidence of plant responses to lunar cycles supports this hypothesis, for which experimental evidence is now anticipated.

HighlightThe long-standing enigma of the triggering of the gregarious flowering of bamboos is possibly solved. Flowering would be inhibited until the occurrence of the species-specific sun-moon phasing. Complete neuroanatomy and sensor maps of dragonfly wings for fly-by-feel monitoring of aeroelasticity Can mechanosensors in animal wings allow reconstruction of the wing aeroelastic states? Little is known about how flying animals utilize wing mechanosensation to monitor the dynamic state of their highly deformable wings. Odonata, dragonflies and damselflies, are a basal lineage of flying insects with excellent flight performance, and their wing mechanics have been studied extensively. Here, we present a comprehensive map of the wing sensory system for two Odonata species, including both the external sensor morphologies and internal neuroanatomy. We identified eight morphological classes of sensors; most were mechanosensors innervated by a single neuron. Their innervation patterns and morphologies minimize axon length and allow morphological latency compensation. We further mapped the major veins of another 13 Odonata species across 10 families and identified consistent sensor distribution patterns, with sensor count scaling with wing length. Finally, we constructed a high-fidelity finite element model of a dragonfly wing for structural analysis. Our dynamic loading simulations revealed features of the strain fields that wing sensor arrays could detect to encode different wing deformation states. Taken together, this work marks the first step toward an integrated understanding of fly-by-feel control in animal flight. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Phenotypic heterogeneity driven by plasticity of the intermediate EMT state governs disease progression and metastasis in breast cancer The Epithelial-to-Mesenchymal Transition (EMT) is a developmental cellular program frequently coopted by cancer cells1 and is a key contributor to both heterogeneity in solid tumors2-4 and later stage chemo-resistance and metastasis5,6. Rather than being a switch from an epithelial to a mesenchymal state, increasing evidence points to the existence of intermediate EMT states, wherein cells co-express both epithelial and mesenchymal traits7-13. Multiple stable intermediate EMT states possessing unique characteristics exist across the EMT spectrum7,8,14,15, contributing to the complex heterogeneity of tumors to promote metastasis16. While much work has been carried out identifying and characterizing EMT-inducing transcription factors17-20, the transcriptional and epigenetic networks responsible for the stability and maintenance of the midpoints along the EMT spectrum are poorly defined. In addition, there are currently no approaches to identifying and quantifying intermediate EMT subpopulations within patient tumors to evaluate their prognostic significance. Using clonally isolated derivatives of the SUM149PT breast cancer cell line, we systematically interrogate how each EMT state independently contributes to heterogeneity and influences metastatic progression, uncovering the role of RUNX2 in stabilizing certain intermediate states. Using SUM149PT-derived tumors as a training set, we develop an entropy-based model to quantify phenotypic heterogeneity and EMT status. Remarkably, the majority of cell states captured in the SUM149PT model are represented in a selection of patient tumors, laying the foundation for quantification of epithelial-mesenchymal heterogeneity and understanding the role of the intermediate EMT state in tumor progression. Electrical Stimulated GLUT4 Signaling Attenuates Critical Illness-Associated Muscle Wasting BackgroundCritical illness myopathy (CIM) is a debilitating condition characterized by the preferential loss of the motor protein myosin. CIM is a byproduct of critical care, attributed to impaired recovery, long-term complications, and mortality. CIM pathophysiology is complex, heterogeneous and remains incompletely understood, however loss of mechanical stimuli contributes to critical illness associated muscle atrophy and weakness. Passive mechanical loading (ML) and electrical stimulation (ES) therapies augment muscle mass and function. While having beneficial outcomes, the mechanistic underpinning of these therapies is less known. Therefore, here we aimed to assess the mechanism by which chronic supramaximal ES ameliorates CIM in a unique experimental rat model of critical care.

MethodsRats were subjected to 8 days critical care conditions entailing deep sedation, controlled mechanical ventilation, and immobilization with and without direct soleus ES. Muscle size and function were assessed at the single cell level. RNAseq and Western blotting were employed to understand the mechanisms driving ES muscle outcomes in CIM.

ResultsFollowing 8 days of controlled mechanical ventilation and immobilization, soleus muscle mass, Myosin:Actin ratio and single muscle fiber maximum force normalized to cross-sectional area (specific force) were reduced by 40-50% (p< 0.0001). ES significantly reduced the loss of soleus muscle fiber cross-sectional area (CSA) and Myosin:Actin ratio by approximately 30% (p< 0.05) yet failed to effect specific force. RNAseq pathway analysis revealed downregulation of insulin signaling in the soleus muscle following critical care and GLUT4 trafficking was reduced by 55% leading to an 85% reduction of muscle glycogen content (p< 0.01). ES promoted phosphofructokinase and insulin signaling pathways to control levels (p< 0.05), consistent with the maintenance of GLUT4 translocation and glycogen levels. AMPK, but not AKT, signaling pathway was stimulated following ES, where the downstream target TBC1D4 increased 3 logFC (p= 0.029) and AMPK-specific P-TBC1D4 levels were increased approximately 2-fold (p= 0.06). Reduction of muscle protein degradation rather than protein synthesis promoted soleus CSA, as ES reduced E3 ubiquitin proteins, Atrogin-1 (p= 0.006) and MuRF1 (p= 0.08) by approximately 50%, downstream of AMPK-FoxO3.

ConclusionsES maintained GLUT4 translocation through increased AMPK-TBC1D4 signaling leading to improved muscle glucose homeostasis. Soleus CSA and myosin content was promoted through reduced protein degradation via AMPK-FoxO3 E3 ligases, Atrogin-1 and MuRF1. These results demonstrate chronic supramaximal ES reduces critical care associated muscle wasting, preserved glucose signaling and reduced muscle protein degradation in CIM. Embryonic exposure to cannabidiol disrupts active neural circuits, an effect increased by Δ9-tetrahydrocannabinol and involving CB1R and CB2R in zebrafish In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development. Remarked Suppression of Aβ42 Protomer-protomer Dissociation Reaction via Pentamer Dimerization Elucidated by Molecular Dynamics Simulation Multimeric protein complexes are molecular apparatuses to regulate biological systems and often determine their fate. Among proteins forming such molecular assemblies, amyloid proteins have drawn attention over a half-century since amyloid fibril formation of these proteins is supposed to be a common pathogenic cause for neurodegenerative diseases. This process is triggered by the accumulation of fibril-like aggregates, while the microscopic mechanisms are mostly elusive due to technical limitation of experimental methodologies in individually observing each of diverse aggregate species in the aqueous solution. We then addressed this problem by employing atomistic molecular dynamics simulations for the paradigmatic amyloid protein, amyloid-{beta} (1-42) (A{beta}42). Seven different dimeric forms of oligomeric A{beta}42 fibril-like aggregate in aqueous solution, ranging from tetramer to decamer, were considered. We found additive effects of the size of these fibril-like aggregates on their thermodynamic stability and have clarified kinetic suppression of protomer-protomer dissociation reactions at and beyond the point of pentamer dimer formation. This observation was obtained from the specific combination of the A{beta}42 protomer structure and the physicochemical condition that we here examined, while it is worthwhile to recall that several amyloid fibrils take dimeric forms of their protomers. We could thus conclude that the stable formation of fibril-like protomer dimer should be involved in a turning point where rapid growth of amyloid fibrils is triggered. Malaria parasites utilize pyrophosphate to fuel an essential proton pump in the ring stage and the transition to trophozoite stage The malaria parasite relies on anaerobic glycolysis for energy supply when growing inside RBCs as its mitochondrion does not produce ATP. The ring stage lasts [~] 22 hours and is traditionally thought to be metabolically quiescent. However, recent studies show that the ring stage is active for several energy-costly processes including gene transcription/translation, protein export, and movement inside the RBC. It has remained unclear if a low glycolytic flux can meet the energy demand of the ring stage. Here we show that the metabolic by-product, pyrophosphate, is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to pump protons across the parasite plasma membrane to maintain the membrane potential and cytosolic pH. Conditional deletion of PfVP1 leads to delayed ring stage development and a complete blockage of the ring-to-trophozoite transition, which can be partially rescued by Arabidopsis thaliana vacuolar pyrophosphatase 1, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae. Proton-pumping pyrophosphatases are absent in humans, which highlights the possibility of developing highly selective VP1 inhibitors against the malaria parasite. Glucose inhibits haemostasis and accelerates diet-induced hyperlipidaemia in zebrafish larvae Hyperglycaemia damages the microvasculature in part through the reduced recruitment of immune cells and interference with platelet signalling, leading to poor wound healing and accelerated lipid deposition in mammals. We investigated the utility of zebrafish larvae to model the effect of glucose on neutrophil and macrophage recruitment to a tail wound, wound-induced haemostasis, and chicken egg yolk feed challenge-induced hyperlipidaemia by supplementing larvae with exogenous glucose by immersion or injection. Neither method of glucose supplementation affected the recruitment of neutrophils and macrophages following tail transection. Glucose injection reduced thrombocyte retention and fibrin plug formation while only thrombocyte retention was reduced by glucose immersion following tail transection. We observed accelerated lipid accumulation in glucose-injected larvae challenged with high fat chicken egg yolk feeding. Our study identifies conserved and divergent effects of high glucose on inflammation, haemostasis, and hyperlipidaemia in zebrafish larvae compared to mammals. Conformist social learning leads to self-organised prevention against adverse bias in risky decision making Given the ubiquity of potentially adverse behavioural bias owing to myopic trial-and-error learning, it seems paradoxical that improvements in decision-making performance through conformist social learning, a process widely considered to be bias amplification, still prevail in animal collective behaviour. Here we show, through model analyses and large-scale interactive behavioural experiments with 585 human subjects, that conformist influence can indeed promote favourable risk taking in repeated experience-based decision making, even though many individuals are systematically biased towards adverse risk aversion. Although strong positive feedback conferred by copying the majoritys behaviour could result in unfavourable informational cascades, our differential equation model of collective behavioural dynamics identified a key role for increasing exploration by negative feedback arising when a weak minority influence undermines the inherent behavioural bias. This  collective behavioural rescue, emerging through coordination of positive and negative feedback, highlights a benefit of collective learning in a broader range of environmental conditions than previously assumed and resolves the ostensible paradox of adaptive collective behavioural flexibility under conformist influences. Immunomodulatory Contribution of Mast Cells to the Regenerative Biomaterial Microenvironment Bioactive immunomodulatory biomaterials have shown promise for influencing the immune response to promote tissue repair and regeneration. Macrophages and T cells have been associated with this response; however, other immune cell types have been traditionally overlooked. In this study, we investigated the role of mast cells in the regulation of the immune response to decellularized biomaterial scaffolds using a subcutaneous implant model. In mast cell-deficient mice, there was dysregulation of the expected M1 to M2 macrophage transition typically induced by the biomaterial scaffold. Polarization progression deviated in a sex specific manner with an early transition to an M2 profile in female mice, while the male response was unable to properly transition past a pro-inflammatory M1 state. Both were reversed with adoptive mast cell transfer. Further investigation of the later stage immune response in male mice determined a greater sustained pro-inflammatory gene expression profile including the IL-1 cytokine family, IL-6, alarmins, and chemokines. These results highlight mast cells as another important cell type that influences the immune response to pro-regenerative biomaterials. Synthetic periphyton as a model system to understand species dynamics in complex microbial freshwater communities Phototrophic biofilms, also known as periphyton, are microbial freshwater communities that drive crucial ecological processes in streams and lakes. Gaining a deep mechanistic understanding of the biological processes occurring in natural periphyton remains challenging due to the high complexity and variability of such communities. To address this challenge, we rationally developed a workflow to construct a synthetic community by co-culturing 26 phototrophic species (i.e., diatoms, green algae and cyanobacteria) that were inoculated in a successional sequence to create a periphytic biofilm on glass slides. We show that this community is diverse, stable and highly reproducible in terms of microbial composition, function and 3D spatial structure of the biofilm. We also demonstrate the ability to monitor microbial dynamics at the single species level during periphyton development and how their abundances are impacted by stressors such as increased temperature and a herbicide, singly and in combination. Overall, such a synthetic periphyton, grown under controlled conditions, can be used as a model system for theory testing through targeted manipulation. A tool for assessing alignment of biomedical data repositories with open, FAIR, citation and trustworthy principles Increasing attention is being paid to the operation of biomedical data repositories in light of efforts to improve how scientific data is handled and made available for the long term. Multiple groups have produced recommendations for functions that biomedical repositories should support, with many using requirements of the FAIR data principles as guidelines. However, FAIR is but one set of principles that has arisen out of the open science community. They are joined by principles governing open science, data citation and trustworthiness, all of which are important aspects for biomedical data repositories to support. Together, these define a framework for data repositories that we call OFCT: Open, FAIR, Citable and Trustworthy. Here we developed an instrument using the open source PolicyModels toolkit that attempts to operationalize key aspects of OFCT principles and piloted the instrument by evaluating eight biomedical community repositories listed by the NIDDK Information Network (dkNET.org). Repositories included both specialist repositories that focused on a particular data type or domain, in this case diabetes and metabolomics, and generalist repositories that accept all data types and domains. The goal of this work was both to obtain a sense of how much the design of current biomedical data repositories align with these principles and to augment the dkNET listing with additional information that may be important to investigators trying to choose a repository, e.g., does the repository fully support data citation? The evaluation was performed from March to November 2020 through inspection of documentation and interaction with the sites by the authors. Overall, although there was little explicit acknowledgement of any of the OFCT principles in our sample, the majority of repositories provided at least some support for their tenets. bulkAnalyseR: An accessible, interactive pipeline for analysing and sharing bulk multi-modal sequencing data Bulk sequencing experiments (single- and multi-omics) are essential for exploring wide-ranging biological questions. To facilitate interactive, exploratory tasks, coupled with the sharing of easily accessible information, we present bulkAnalyseR, a package integrating state-of-the-art approaches using an expression matrix as the starting point (pre-processing functions are available as part of the package). Static summary images are replaced with interactive panels illustrating quality-checking, differential expression analysis (with noise detection) and biological interpretation (enrichment analyses, identification expression patterns, followed by inference and comparison of regulatory interactions). bulkAnalyseR can handle different modalities, facilitating robust integration and comparison of cis-, trans- and customised regulatory networks.

bulkAnalyseR is available on CRAN and GitHub, with extensive documentation and usage examples (https://github.com/Core-Bioinformatics/bulkAnalyseR, https://cran.r-project.org/web/packages/bulkAnalyseR/)

ContactIrina Mohorianu iim22@cam.ac.uk Age-associated B cells are long-lasting effectors that restrain reactivation of latent γHV68 Age-associated B cells (ABCs; CD19+CD11c+T-bet+) are a unique population that are increased in an array of viral infections, though their role during latent infection is largely unexplored. Here, we use murine gammaherpesvirus 68 ({gamma}HV68) to demonstrate that ABCs remain elevated long-term during latent infection and express IFN{gamma} and TNF. Using a strain of {gamma}HV68 that is cleared following acute infection, we show that ABCs persist in the absence of latent virus, though their expression of IFN{gamma} and TNF is decreased. With a fluorescent virus we demonstrate that ABCs are infected with {gamma}HV68 at similar rates to other previously activated B cells. We find that mice without ABCs display defects in anti-viral IgG2a/c antibodies and are more susceptible to {gamma}HV68 reactivation when challenged with heterologous infection. Together, these results indicate that ABCs are a persistent effector subset during latent viral infection that restrains {gamma}HV68 reactivation. Bespoke data augmentation and network construction enable developmental morphological classification on limited microscopy datasets Deep learning is a powerful tool for image classification, yet training deep learning models often requires large datasets, which are not always available in biological research. Here we use a small dataset to train an accurate classifier by employing data augmentation regimes and Bayesian optimisation. We establish a new staging system for the Hamburger-Hamilton stage 10 chick embryo brain, based on morphology, identifying three sub-stages uncoupled from somite number, the usual metric used to stage chick embryos. Incorporating biologically informed, data-driven preprocessing, we train a deep convolutional neural network to classify our sub-stages. We find that augmenting our images with a combination of rotation, blur, shear, and cutout transformations is effective in training an accurate classifier on a small microscopy dataset. We then apply our approach to an unrelated small microscopy dataset comprising images of limb buds, finding good agreement with the results from brain classification. Through saliency analysis, we ensure that relevant features are being used by our classifier, gain insight into the efficacy of different data augmentations, and identify sub-stage specific features. In summary, we apply biological domain expertise to design effective data augmentation regimes for training a neural network-based image classifier on small, specialised datasets, classifying the developing brains and limbs with up to 90.9% and 94.4% accuracy respectively. These reliable classifiers can inform future experiments. Visual gene expression reveals a cone to rod developmental progression in deep-sea fishes Vertebrates use cone cells in the retina for colour vision and rod cells to see in dim light. Many deep-sea fishes have adapted to their environment to have only rod cells in the retina, while both rod and cone genes are still preserved in their genomes. As deep-sea fish larvae start their lives in the shallow, and only later submerge to the depth, they have to cope with diverse environmental conditions during ontogeny. Using a comparative transcriptomic approach in 20 deep-sea fish species from eight teleost orders, we report on a developmental cone-to-rod switch. While adults mostly rely on rod opsin (RH1) for vision in dim light, larvae almost exclusively express middle-wavelength-sensitive ("green") cone opsins (RH2) in their retinas. The phototransduction cascade genes follow a similar ontogenetic pattern of cone-followed by rod-specific gene expression in most species, except for the pearleye and sabretooth (Aulopiformes), in which the cone cascade remains dominant throughout development. By inspecting the whole genomes of five deep-sea species (four of them sequenced within this study: Idiacanthus fasciola, Chauliodus sloani; Stomiiformes; Coccorella atlantica, and Scopelarchus michaelsarsi; Aulopiformes), we found that deep-sea fish possess one or two copies of the rod RH1 opsin gene, and up to seven copies of the cone RH2 opsin genes in their genomes, while other cone opsin classes have been mostly lost. Our findings hence provide molecular evidence for a limited opsin gene repertoire and a conserved vertebrate pattern whereby cone photoreceptors develop first and rod photoreceptors are added only at later developmental stages. Genome archaeology of two laboratory Salmonella enterica enterica sv Typhimurium The Salmonella research community has used strains and bacteriophages over decades, exchanging useful new isolates among laboratories for study of cell surface antigens, metabolic pathways and restriction-modification studies. Here we present the sequences of two laboratory Salmonella strains (STK005, an isolate of LB5000; and its descendant ER3625). In the ancestry of LB5000, segments of ~15 and ~42 kb were introduced from Salmonella enterica sv Abony 103 into Salmonella enterica sv Typhimurium LT2, forming strain SD14; this strain is thus a hybrid of S. enterica isolates. Strains in the SD14 lineage were used to define flagellar antigens from the 1950s to the 1970s, and to define three restriction-modification systems from the 1960s to the 1980s. LB5000 was also used as host in phage typing systems used by epidemiologists. In the age of cheaper and easier sequencing, this resource will provide access to the sequence that underlies the extensive literature.