Fungicide resistance characterised across seven modes of action in a Botrytis cinerea isolated from Australian vineyards BACKGROUNDBotrytis bunch rot, caused by Botrytis cinerea, is an economically important disease of grapes in Australia and across grape growing regions worldwide. Control of this disease relies on canopy management and the application of fungicides. Fungicide application can lead to the selection of resistant B. cinerea populations, which has an adverse effect on management of the disease. Characterising the distribution and severity of resistant B. cinerea populations is needed to inform resistance management strategies.

RESULTSIn this study, 725 isolates were sampled from 75 Australian vineyards during 2013 - 2016 and were screened against seven fungicides with different modes of action (MOAs). The resistance frequencies for azoxystrobin, boscalid, fenhexamid, fludioxonil, iprodione, pyrimethanil and tebuconazole were 5, 2.8, 2.1, 6.2, 11.6, 7.7 and 2.9% respectively. Nearly half of the resistant isolates (43.8%) were resistant to more than one of the fungicides tested. The frequency of vineyards with at least one isolate simultaneously resistant to 1, 2, 3, 4 or 5 fungicides was 19.5, 7.8, 6.5, 10.4 and 2.6%. Resistance was associated with previously published genotypes in CytB (G143A), SdhB (H272R/Y), Erg27 (F412S), Mrr1 (D354Y), Bos1 (I365S, N373S + Q369P, I365S + D757N) and Pos5 (V273I, P319A, L412F/V). Novel genotypes were also described in Mrr1 (S611N, D616G) Pos5 (V273L) and Cyp51 (P347S). Expression analysis was used to characterise fludioxonil resistant isolates exhibiting overexpression (6.3-9.6-fold) of the ABC transporter gene AtrB (MDR1 phenotype).

CONCLUSIONResistance frequencies were lower when compared to most previously published surveys of B. cinerea resistance in grape and other crops. Nevertheless, continued monitoring of critical MOAs used in Australian vineyards is recommended.
Nrg1/ErbB Signaling-Mediated Regulation of Fibrosis After Myocardial Infarction RATIONALEAppropriate fibrotic tissue formation after myocardial infarction (MI) is crucial to maintenance of the hearts structure. Reparative or M2-like macrophages play a vital role in post-MI fibrosis by activating cardiac fibroblasts. However, the mechanism by which post-MI cardiac fibrosis is regulated is not fully understood.

OBJECTIVEWe investigated the cellular and molecular mechanisms of post-MI fibrotic tissue formation, especially those related to regulation of cellular senescence and apoptosis.

METHODS AND RESULTSIn vivo and in vitro experiments were used to investigate the molecular and cellular mechanisms through which post-MI fibrosis occurs, with a focus on the role of M2-like macrophages. Microarray analysis revealed that CD206+F4/80+CD11b+ M2-like macrophages collected from mouse hearts on post-MI day 7 showed increased expression of neuregulin 1 (Nrg1). Nrg1 receptor epidermal growth factor receptor ErbB was expressed on cardiac fibroblasts in the infarct area. In cardiac fibroblasts in which hydrogen peroxide-induced senescence, M2-like macrophage-derived Nrg1 suppressed both senescence and apoptosis of the fibroblasts, whereas blockade of ErbB function significantly accelerated. M2-like macrophage-derived Nrg1/ErbB/PI3K/Akt signaling, which was shown to be related to anti-senescence, was activated in damaged cardiac fibroblasts. Interestingly, systemic blockade of ErbB function in MI model mice enhanced senescence and apoptosis of cardiac fibroblasts and exacerbated inflammation. Further, increased accumulation of M2-like macrophages resulted in excessive progression of fibrosis in post-MI murine hearts. The molecular mechanism underlying regulation of fibrotic tissue formation in the infarcted myocardium was shown in part to be attenuation of apoptosis and senescence of cardiac fibroblasts by activation of Nrg1/ErbB/PI3K/Akt signaling.

CONCLUSIONSM2-like macrophage-mediated regulation of Nrg1/ErbB signaling, have a substantial effect on fibrotic tissue formation in the infarcted adult mouse heart, is critical for suppressing the progression of senescence and apoptosis of cardiac fibroblasts.
Protective host-dependent antagonism among 	Pseudomonas in the Arabidopsis phyllosphere The plant microbiome is a rich biotic environment, comprising numerous taxa. The community structure of these colonizers is constrained by multiple factors, including host-microbe and microbe-microbe interactions, as well as the interplay between the two. While much can be learned from pairwise relationships between individual hosts and microbes, or individual microbes with themselves, the ensemble of interrelations between the host and microbial consortia may lead to different outcomes that are not easily predicted from the individual interactions. Their study can thus provide new insights into the complex relationship between plants and microbes. Of particular importance is how strain-specific such plant-microbe-microbe interactions are, and how they eventually affect plant health. Here, we test strain-level interactions in the phyllosphere between groups of co-existing commensal and pathogenic Pseudomonas among each other and with A. thaliana, by employing synthetic communities of genome-barcoded isolates. We found that commensal Pseudomonas prompted a host response leading to a selective inhibition of a specific pathogenic lineage, resulting in plant protection. The extent of plant protection, however, was dependent on plant genotype, indicating that these effects are host-mediated. There were similar genotype-specific effects on the microbe side, as we could pinpoint an individual Pseudomonas isolate as the predominant cause for this differential interaction. Collectively, our work highlights how within-species genetic differences on both the host and microbe side can have profound effects on host-microbe-microbe dynamics. The paradigm that we have established provides a platform for the study of host-dependent microbe-microbe competition and cooperation in the A. thaliana-Pseudomonas system. Extending and using anatomical vocabularies in the Stimulating Peripheral Activity to Relieve Conditions (SPARC) program The Stimulating Peripheral Activity to Relieve Conditions (SPARC) program is a US National Institutes of Health-funded effort to improve our understanding of the neural circuitry of the autonomic nervous system in support of bioelectronic medicine. As part of this effort, the SPARC program is generating multi-species, multimodal data, models, simulations, and anatomical maps supported by a comprehensive knowledge base of autonomic circuitry. To facilitate the organization of and integration across multi-faceted SPARC data and models, SPARC is implementing the FAIR data principles to ensure that all SPARC products are findable, accessible, interoperable, and reusable. We are therefore annotating and describing all products with a common FAIR vocabulary. The SPARC Vocabulary is built from a set of community ontologies covering major domains relevant to SPARC, including anatomy, physiology, experimental techniques, and molecules. The SPARC Vocabulary is incorporated into tools researchers use to segment and annotate their data, facilitating the application of these ontologies for annotation of research data. However, since investigators perform deep annotations on experimental data, not all terms and relationships are available in community ontologies. We therefore implemented a term management and vocabulary extension pipeline where SPARC researchers may extend the SPARC Vocabulary using InterLex, an online vocabulary management system. To ensure the quality of contributed terms, we have set up a curated term request and review pipeline specifically for anatomical terms involving expert review. Accepted terms are added to the SPARC Vocabulary and, when appropriate, contributed back to community ontologies to enhance autonomic nervous system coverage. Here, we provide an overview of the SPARC Vocabulary, the infrastructure and process for implementing the term management and review pipeline. In an analysis of > 300 anatomical contributed terms, the majority represented composite terms that necessitated combining terms within and across existing ontologies. Although these terms are not good candidates for community ontologies, they can be linked to structures contained within these ontologies. We conclude that the term request pipeline serves as a useful adjunct to community ontologies for annotating experimental data and increases the FAIRness of SPARC data. The unfolded protein response triggers the immune deficiency pathway in ticks The insect immune deficiency (IMD) pathway is a defense mechanism that senses and responds to Gram negative bacteria. Ticks lack genes encoding upstream components that initiate the IMD pathway. Despite this deficiency, core signaling molecules are present and functionally restrict tick-borne pathogens. The molecular events preceding activation remain undefined. Here, we show that the Unfolded Protein Response (UPR) initiates the IMD network in Ixodes scapularis ticks. The endoplasmic reticulum (ER) stress receptor, IRE1, is phosphorylated in response to tick-borne bacteria, but does not splice the mRNA encoding XBP1. Instead, through protein modeling and reciprocal pulldowns, we show that Ixodes IRE1 complexes with TRAF2. Disrupting IRE1-TRAF2 signaling blocks IMD pathway activation and diminishes the production of reactive oxygen species. Through in vitro, in vivo, and ex vivo techniques we demonstrate that the UPR-IMD pathway circuitry limits the Lyme disease-causing spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale (anaplasmosis). Altogether, our study uncovers a novel linkage between the UPR and the IMD pathway in ticks. Efficient and Economical Targeted Insertion in Plant Genomes via Protoplast Regeneration Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50.0% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60-nt donor containing 27 nt in each homologous arm, 6 of 22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6-bp HindIII site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants. Environment dependent benefits of inter-individual variation in honey bee recruitment Inter-individual differences in behaviour within the members of a social group can affect the groups productivity. In eusocial insects, individual differences amongst workers in a colony play a central role division of labour and task allocation. Extensive empirical and theoretical work has highlighted variation in response thresholds as a proximate mechanism underlying individual behavioural differences and hence division of labour. However, other response parameters, like response probability and intensity, can affect these differences. In this study, we first extended a previously published agent-based model on honey bee foraging to understand the relative importance of response (dance) probability and response (dance) intensity in the task of recruitment. Comparing variation obtained from the simulations with previously published empirical data, we found that response intensity plays a more important role than probability in producing consistent inter-individual differences in recruitment behaviour. We then explored the benefits provided by this individual variation in recruitment behaviour to the colonys collective foraging effort under different environmental conditions. Our results revealed that individual variation leads to a greater energetic yield per forager, but only when food is abundant. Our study highlights the need to consider all response parameters while studying division of labour and adds to the growing body of evidence linking individual variation in behavioural responses to the success of social groups. IRGM/Irgm1 Aggravates Progression of Atherosclerosis by Inducing Macrophage Apoptosis through the MAPK Signaling Pathway AimsAtherosclerosis underlies most cardiovascular diseases, among which acute coronary syndrome (ACS) caused by plaque rupture (PR) often leads to death. Immune-related GTPases (IRGM/Irgm1) have been extensively studied in inflammatory diseases, but their role in atherosclerosis is unclear. Determining how IRGM/Irgm1 promotes atherosclerotic plaque vulnerability will provide information for new biomarkers and/or therapeutic targets.

Methods and resultsWe identified ruptured and unruptured plaques by optical coherence tomography, and found that serum IRGM was highly expressed in patients with ST-segment elevation myocardial infarction in PR. We used ApoE-/-Irgm1+/+, ApoE-/-Irgm1+/- mice and chimeric mice to establish a model of advanced atherosclerosis. The results of pathological experiments showed that Irgm1 caused plaque necrosis. The ratio of neutral lipids and cholesterol crystals increases, while the content of collagen fibers decreases, aggravating the destabilization of atherosclerotic plaques. In vitro, we used multiple approaches to confirm that Irgm1 promotes macrophage apoptosis by promoting the production of reactive oxygen species and activating the MAPK signaling pathway.

ConclusionsIRGM may be a potential risk factor for PR. Mechanistic studies have shown that IRGM/Irgm1 contributes to the formation and rupture of fragile plaques. This is partly mediated by the induction of macrophage apoptosis via the MAPK signaling pathway. IRGM may offer new strategies for early treatment of ACS.

Translation viewOur findings indicate that IRGM/Irgm1 contributes to formation and rupture of vulnerable plaques. It suggests that IRGM may provide a new target for the early treatment of ACS. Single-cell Transcriptomic Profiling Unveils Cardiac Cell-type Specific Response to Maternal Hyperglycemia Underlying the Risk of Congenital Heart Defects Congenital heart disease (CHD) is the most prevalent structural malformations of the heart affecting [~]1% of live births. To date, both damaging genetic variations and adverse environmental exposure such as maternal diabetes have been found to cause CHD. Clinical studies show [~]fivefold higher risk of CHD in the offspring of mothers with pregestational diabetes. Maternal pregestational diabetes affects the gene regulatory networks key to proper cardiac development in the fetus. However, the cell-type specificity of these gene regulatory responses to maternal diabetes and their association with the observed cardiac defects in the fetuses remains unknown. To uncover the transcriptional responses to maternal diabetes in the early embryonic heart, we used an established murine model of pregestational diabetes. In this model, we have previously demonstrated an increased incidence of CHD. Here, we show maternal hyperglycemia (matHG) elicits diverse cellular responses during heart development by single-cell RNA-sequencing in embryonic hearts exposed to control and matHG environment. Through differential gene-expression and pseudotime trajectory analyses of this data, we identified changes in lineage specifying transcription factors, predominantly affecting Isl1+ second heart field progenitors and Tnnt2+ cardiomyocytes with matHG. Using in vivo cell-lineage tracing studies, we confirmed that matHG exposure leads to impaired second heart field-derived cardiomyocyte differentiation. Finally, this work identifies matHG-mediated transcriptional determinants in cardiac cell lineages elevate CHD risk and show perturbations in Isl1-dependent gene-regulatory network (Isl1-GRN) affect cardiomyocyte differentiation. Functional analysis of this GRN in cardiac progenitor cells will provide further mechanistic insights into matHG-induced severity of CHD associated with diabetic pregnancies. Evolutionary association of receptor-wide amino acids with G protein coupling selectivity in aminergic GPCRs G protein-coupled receptors (GPCRs) induce signal transduction pathways through coupling to four main subtypes of G proteins (Gs, Gi, Gq, G12/13), selectively. However, G protein selective activation mechanisms and residual determinants in GPCRs have remained obscure. Herein, we performed an extensive phylogenetic analysis and identified specifically conserved residues for the receptors having similar coupling profiles in each aminergic receptor. By integrating our methodology of differential evolutionary conservation of G protein-specific amino acids with structural analyses, we identified selective activation networks for Gs, Gi1, Go, and Gq. To validate that these networks could determine coupling selectivity we further analyzed Gs specific activation network and associated it with the larger TM6 tilt which is a signature of Gs-coupled receptors. Through molecular dynamics simulations, we showed that previously uncharacterized Glycine at position 7x41 plays an important role in both receptor activation and Gs coupling selectivity by inducing a larger TM6 movement. Finally, we gathered our results into a comprehensive model of G protein selectivity called "sequential switches of activation" describing three main molecular switches controlling GPCR activation: ligand binding, G protein selective activation mechanisms and G protein contact. Visual Clustering of Transcriptomic Data from Primary and Metastatic Tumors - Dependencies and Novel Pitfalls Personalized Oncology is a rapidly evolving area and offers cancer patients therapy options more specific than ever. Yet, there is still a lack of understanding regarding transcriptomic similarities or differences of metastases and corresponding primary sites. Applying two unsupervised dimension reduction methods (t-Distributed Stochastic Neighbor Embedding (t-SNE) and Uniform Manifold Approximation Projection (UMAP)) on three datasets of metastases (n=682 samples) with three different data transformations (unprocessed, log10 as well as log10+1 transformed values), we visualized potential underlying clusters. Additionally, we analyzed two datasets (n=616 samples) containing metastases and primary tumors of one entity, to point out potential familiarities. Using these methods, no tight link between site of resection and cluster formation outcome could be demonstrated, neither for datasets consisting of solely metastasis nor mixed datasets. Instead, dimension reduction methods and data transformation significantly impacted visual clustering results. Our findings strongly suggest data transformation to be considered as another key element in interpretation of visual clustering approaches along with initialization and different parameters. Furthermore, the results indicate only minor transcriptional differences for metastases and corresponding primary tumors. ZIKV disrupts placental ultrastructure and drug transporter expression in mice Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome. ElectroEncephaloGraphy robust linear modelling using weights reflecting single trials' dynamics Being able to remove or weigh down the influence of outlier data is desirable for any statistical models. While Magnetic and ElectroEncephaloGraphic (MEEG) data are often averaged across trials per condition, it is becoming common practice to use information from all trials to build statistical linear models. Individual trials can, however, have considerable weight and thus bias inferential results (effect sizes as well as thresholded t/F/p maps). Here, rather than looking for univariate outliers, defined independently at each measurement point, we apply the principal component projection (PCP) method at each channel, deriving a single weight per trial at each channel independently. Using both synthetic data and open EEG data, we show (1) that PCP is efficient at detecting a large variety of outlying trials; (2) how PCP-based weights can be implemented in the context of the general linear model (GLM) with accurate control of type 1 family-wise error rate; and (3) that our PCP-based Weighted Least Square (WLS) approach increases the statistical power of group analyses as well as a much slower Iterative Reweighted Least Squares (IRLS), although the weighting scheme is markedly different. Together, our results show that WLS based on PCP weights derived from whole trial profiles is an efficient method to weigh down the influence of outlier EEG data in linear models.

Data availabilityall data used are publicly available (CC0), all code (simulations and data analyzes) is also available online in the LIMO MEEG GitHub repository (MIT license). Inhibitors of ERp44, PDIA1, and AGR2 induce disulfide-mediated oligomerization of Death Receptors 4 and 5 and cancer cell death Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8. Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression BackgroundChimeric antigen receptor (CAR) T cells traditionally harbor viral vector-based sequences that encode the CAR transgene in the genome. These T cell products have yet to show consistent anti-tumor activity in patients with solid tumors. Further, viral vector manufacturing is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures.

MethodsAnti-GD2 CAR T cells were generated using CRISPR/Cas9 within nine days using recombinant Cas9 protein and nucleic acids, without any viral vectors or animal components. The CAR was specifically targeted to the T Cell Receptor Alpha Constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, scRNA-seq, spectral cytometry, and ELISA assays. Functionality was evaluated in vivo in an NSG xenograft neuroblastoma model.

ResultsIn comparison to traditional retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory phenotype, and low tonic signaling prior to infusion arising in part from the the knockout of the TCR. Upon exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibited specific cytotoxicity against GD2+ cells in vitro and induced solid tumor regression in vivo, with robust homing, persistence, and low exhaustion against a human neuroblastoma xenograft model.

ConclusionsThis proof-of-principle study leveraging virus-free genome editing technology could enable flexible manufacturing of clinically relevant, high-quality CAR T cells to treat cancers, including solid tumors. Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, "aphaenogastroid" body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a "pheidoloid" body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25{degrees}C. We discuss possible functions of the tubercles of queen larvae based on previous studies. Functional buffering via cell-specific gene expression promotes tissue homeostasis and cancer robustness Functional buffering that ensures biological robustness is critical for maintaining tissue homeostasis, organismal survival, and evolution of novelty. However, the mechanism underlying functional buffering, particularly in multicellular organisms, remains largely elusive. Here, we developed an inference index (C-score) for Cell-specific Expression- BUffering (CEBU), whereby functional buffering is mediated via expression of buffering genes in specific cells and tissues in humans. By computing C-scores across 684 human cell lines using genome-wide CRISPR screens and transcriptomic RNA-seq, we report that C- score-identified putative buffering gene pairs are enriched for members of the same duplicated gene family, pathway, and protein complex. Furthermore, CEBU is especially prevalent in tissues of low regenerative capacity (e.g., bone and neuronal tissues) and is weakest in highly regenerative blood cells, linking functional buffering to tissue regeneration. Clinically, the buffering capacity enabled by CEBU can help predict patient survival for multiple cancers. Our results reveal CEBU as a buffering mechanism contributing to tissue homeostasis and cancer robustness in humans.

Summary blurbWe unveil a genome-wide functional buffering mechanism, termed Cell-specific Expression Buffering (CEBU), whereby gene expression contributes to functional buffering in specific cell types and tissues. We link CEBU to genetic interactions, tissue homeostasis and cancer robustness. De novo chromosome-length assembly of the mule deer (Odocoileus hemionus) genome The mule deer (Odocoileus hemionus) is an ungulate species that ranges from western Canada to central Mexico. Mule deer are an essential source of food for many predators, are relatively abundant, and commonly make broad migration movements. A clearer understanding of the mule deer genome can help facilitate knowledge of its population genetics, movements, and demographic history, aiding in conservation efforts. While mule deer are excellent candidates for population genomic studies because of their large population size, continuous distribution, and diversity of habitat, few genomic resources are currently available for this species. Here, we sequence and assemble the mule deer genome into a highly contiguous chromosome-length assembly for use in future research using long-read sequencing and Hi-C. We also provide a genome annotation and compare demographic histories of the mule deer and white-tail deer using PSMC. We expect this assembly to be a valuable resource in the continued study and conservation of mule deer. Light activation of Orange Carotenoid Protein reveals initial C8'-C7' double bond trans/cis isomerization Orange Carotenoid protein (OCP) is the only known photoreceptor which uses carotenoid for its activation1,2. It is found exclusively in cyanobacteria, where it functions to control light-harvesting of the photosynthetic machinery. However, the photochemical reactions and structural dynamics of this unique photosensing process are not yet resolved. We present time-resolved crystal structures at second-to-minute delays under bright illumination, capturing the early photoproduct and structures of the subsequent reaction intermediates. The first stable photoproduct shows carotenoid trans/cis isomerization at the C7-C8double bond and structural changes in the N-terminal domain with minute timescale kinetics. These are followed by a thermally-driven cis/trans isomerization that recovers to the dark state carotenoid configuration. Structural changes propagate to the C-terminal domain, resulting, at later time, in the H-bond rupture of the carotenoid keto group with protein residues. The isomerization and its transient nature are confirmed in OCP crystals and solution by FTIR and UV/Vis spectroscopy. This study reveals the isomerization of the carotenoid and subsequent thermal structural reactions as the basis of OCP photoreception. Understanding and potentially controlling the OCP dynamics offers the prospect of novel applications in biomass engineering3 as well as in optogenetics and bioimaging. Protein-protein interactions on membrane surfaces analysed using pull-downs with supported bilayers on silica beads Discovery-based proteomics workflows that identify novel interactors rely on immunoprecipitations or pull-downs with genetically-tagged bait proteins immobilized on appropriate matrices. But strategies to analyse protein interactions on a diffusible membrane surface combined with the practical ease of pull-downs remain unavailable. Such strategies are important to analyse protein complexes that mature in composition and stability because of diffusion-based encounter between participant proteins. Here, we describe a generic pull-down strategy to analyse such complexes using chelating lipid-containing supported bilayers formed on silica beads. These templates can display desired His-tagged bait proteins on a diffusible membrane surface. Using clathrin-mediated endocytosis as a paradigm, we find that the clathrin-binding adaptor protein epsin1 displayed as bait on these templates pulls down significantly higher amounts of clathrin from brain lysates than when immobilized on conventional matrices. Together, our results establish the potential of such templates as superior matrices for analysing protein-protein interactions and resultant complexes formed on membrane surfaces. 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. Host species identity shapes the diversity and structure of insect microbiota As for most of the life that inhabits our planet, microorganisms play an essential role in the fitness of insects, including nutrition, reproduction, defence, and many other functions. More recently, we assisted in an exponential growth of studies describing the taxonomical composition of bacterial communities across insects phylogeny. However, there is still an outstanding question that needs to be answered: which factors contribute most to shape insects microbiomes? This study tries to find an answer to this question by taking advantage of publicly available sequencing data and reanalysing over 4,000 samples of insect-associated bacterial communities under a common framework. Results suggest that insect taxonomy has a wider impact on the structure and diversity of their associated microbial communities than the other factors considered (diet, sex, life stage, sample origin, and treatment). However, when specifically testing for signatures of co-diversification of insect species and their microbiota, analyses found weak support for this, suggesting that while insect species strongly drive the structure and diversity of insect microbiota, the diversification of those microbial communities did not follow their hosts phylogeny. Furthermore, a parallel survey of the literature highlights several methodological limitations that need to be considered in future research endeavours. 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. Macroscopic control of synchronous electrical signaling with chemically-excited gene expression Excitable cells can convert electrical signals into chemical outputs to facilitate the active transport of information across larger distances. This electrical-to-chemical conversion requires a tightly regulated expression of ion channels. Alterations of ion channel expression provide landmarks of numerous pathological diseases, such as cardiac arrhythmia, epilepsy, or cancer. Although the activity of ion channels can be locally regulated by external light or chemical stimulus, it remains challenging to coordinate the expression of ion channels on extended spatial-temporal scales in a non-invasive manner. Here, we have engineered yeast S. cerevisiae to read and convert local chemical concentrations into a dynamic electrical field distributed across cell populations. The core mechanism encodes a chemically-excitable dual-feedback gene circuit that precisely tunes the expression domain of potassium channels, globally coordinating cyclic firing of the plasma membrane potential (PMP). We demonstrate that this mechanism leverages an engineered constitutively open bacterial potassium channel KcsA to directly couple chemical stimuli with ion flux through gene expression and it can interface with the host ion channels through the pulsatile production of toxins. Our study provides a robust synthetic transcriptional toolbox underlying the conversion of local chemical environments into spatiotemporally organized electrical impulses for various cellular engineering, synthetic biology, and potential therapeutic applications. Bacterial community dynamics during embryonic development of the little skate (Leucoraja erinacea) BackgroundMicrobial transmission from parent to offspring is hypothesized to be widespread in vertebrates. However, evidence for this is limited as many evolutionarily important clades remain unexamined. There is currently no data on the microbiota associated with any Chondrichthyan species during embryonic development, despite the global distribution, ecological importance, and phylogenetic position of this clade. In this study, we take the first steps towards filling this gap by investigating the microbiota associated with embryonic development in the little skate, Leucoraja erinacea, a common North Atlantic species and popular system for chondrichthyan biology.

MethodsTo assess the potential for bacterial transmission in an oviparous chondrichthyan, we used 16S rRNA amplicon sequencing to characterize the microbial communities associated with the skin, gill, and egg capsule of the little skate, at six points during ontogeny. Community composition was analyzed using the QIIME2 pipeline and microbial continuity between stages was tracked using FEAST.

ResultsWe identify site-specific and stage-specific microbiota dominated by the bacterial phyla Proteobacteria and Bacteroidetes. This composition is similar to, but distinct from, that of previously published data on the adult microbiota of other chondrichthyan species. Our data reveal that the skate egg capsule harbors a highly diverse bacterial community-particularly on the internal surface of the capsule-and facilitates intergenerational microbial transfer to the offspring. Embryonic skin and external gill tissues host similar bacterial communities; the skin and gill communities later diverge as the internal gills and skin denticles develop.

ConclusionsOur study is the first exploration of the chondrichthyan microbiota throughout ontogeny and provides the first evidence of vertical transmission in this group. BI-1 mediated cascade improves redox homeostasis during thermal stress and prevents oxidative damage in a preconditioned stony coral Global coral reef decline is largely driven by the breakdown of the coral-algal symbiosis during temperature stress. Corals can acclimatize to higher temperatures, but the cellular processes underlying this ability are poorly understood. We show that preconditioning-based improvements in thermal tolerance in Pocillopora acuta are accompanied by increases in host glutathione reductase (GR) activity and expression, which prevents DNA damage. A strong correlation between GR and BI-1 expression in heat-stressed preconditioned corals and the presence of an antioxidant response element (ARE) in the GR promoter suggest BI-1 could regulate GR expression through Nrf2/ARE pathway. To fortify this link, we developed an siRNA- mediated gene knockdown protocol and targeted the coral BI-1 gene. BI-1 knock-down specifically decreased GR expression and activity and increased oxidative DNA damage in heat- stressed preconditioned corals, showing that BI-1-mediated enhanced antioxidant response during acute heat stress is a key mechanism that prevents oxidative DNA damage after preconditioning.

TeaserPreconditioning improves redox homeostasis and prevents oxidative stress in a thermally stressed reef-building coral