Curation, inference, and assessment of a globally reconstructed gene regulatory network for Streptomyces coelicolor Streptomyces coelicolor A3(2) is a model microorganism for the study of Streptomycetes, antibiotic production, and secondary metabolism in general. Even though S. coelicolor has an outstanding variety of regulators among bacteria, little effort to globally study its transcription has been made. We manually curated 29 years of literature and databases to assemble a meta-curated experimentally-validated gene regulatory network (GRN) with 5386 genes and 9707 regulatory interactions ([~]41% of the total expected interactions). This provides the most extensive and up-to-date reconstruction available for the regulatory circuitry of this organism. Only [~]6% (534/9707) are supported by experiments confirming the binding of the transcription factor to the upstream region of the target gene, the so-called "strong" evidence. While for the remaining interactions there is no confirmation of direct binding. To tackle network incompleteness, we performed network inference using several methods (including two proposed here) for motif identification in DNA sequences and GRN inference from transcriptomics. Further, we contrasted the structural properties and functional architecture of the networks to assess the reliability of the predictions, finding the inference from DNA sequence data to be the most trustworthy approach. Finally, we show two applications of the inferred and the curated networks. The inference allowed us to propose novel transcription factors for the key Streptomyces antibiotic regulatory proteins (SARPs). The curated network allowed us to study the conservation of the system-level components between S. coelicolor and Corynebacterium glutamicum. There we identified the basal machinery as the common signature between the two organisms. The curated networks were deposited in Abasy Atlas (https://abasy.ccg.unam.mx/) while the inferences are available as Supplementary Material.
Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software ASMv1.0 Open reading frame (ORF) variant libraries have advanced our ability to query the functions of a large number of variants of a protein simultaneously in a single experiment. Variant libraries targeting full-length ORFs typically consists of all possible single-amino-acid substitutions and a stop codon at each amino-acid position. Because a variant differs from the template ORF by a single codon variation, variant quantification presents the most profound challenge to this technology. Efforts such as dividing a library into sub-libraries for direct sequencing, or tag-directed subassembly are practical only for small ORFs. Our approach, however, features an enhanced variant-calling tool that processes massive shotgun sequencing data, allowing one to screen a single variant library for ORFs sized up to [~]3600 bases. The variant-calling tool detects variants reliably, and also presents variant annotations in datafiles enabling analyses that have reshaped our strategies governing library design, screen deconvolution, sequencing and sequencing data analysis.
Metacommunity dynamics and the detection of species associations in co-occurrence analyses: why patch disturbance matters O_LI1: Many statistical methods attempt to detect species associations - and so infer inter-specific interactions - from species co-occurrence patterns. Habitat heterogeneity and out-of-equilibrium colonization histories are well recognized as potentially causing species associations, even when interactions are absent. The potential for patch disturbance, a classical component of metacommunity dynamics, to also drive spurious species associations has however been over-looked.
C_LIO_LI2: Using a new general metacommunity model, we derive mathematical predictions regarding how patch disturbance would affect the patterns of species associations detected in "null" co-occurrence matrices. We also conduct numerical simulations to test our predictions and to compare the performance of several widespread statistical methods, including direct tests of pairwise independence, matrix permutation approaches and joint species distribution modelling.
C_LIO_LI3: We show how classical metacommunity dynamics can produce statistical associations, both positive and negative, even when species do not interact, when there is no habitat heterogeneity, and at equilibrium. This occurs as soon as there is some rate of patch disturbance (i.e. simultaneous extinction of several species in a patch) and/or a finite life-span of patches, a common feature of a broad range of plant, animal or microbial systems.
C_LIO_LI4: Patch disturbance can compromise species co-occurrence analyses and cause the artefactual detection of species associations if not taken into account. Including patch age (i.e. the time since the last patch disturbance event) as a covariate in a joint species distribution model can resolve the artefact. However, this requires additional data that often are not available in practice. We argue that the consequences of patch disturbance should not be underestimated when analysing species distribution patterns in metacommunity-like systems.
C_LI Marburg and Ebola virus infections elicit a complex, muted inflammatory state in bats The Marburg and Ebola filoviruses cause a severe, often fatal, disease in humans and nonhuman primates but have only subclinical effects in bats, including Egyptian rousettes, which are a natural reservoir of Marburg virus. A fundamental question is why these viruses are highly pathogenic in humans but fail to cause disease in bats. To understand how bats resist the disease caused by filoviruses, we infected one cohort of Egyptian rousette bats with Marburg virus and another cohort with Ebola virus and harvested multiple tissues for mRNA expression analysis. While virus transcripts were found primarily in the liver, Principal component analysis (PCA) revealed coordinated changes across multiple tissues. Gene signatures in kidney and liver pointed at induction of vasodilation, reduction of coagulation and changes in the regulation of iron metabolism. Signatures of immune response detected in spleen and liver indicated a robust anti-inflammatory state signified by macrophages in the M2 state and an active T cell response. Many of the responsive genes were found to be evolutionarily divergent, providing a framework for understanding the differences in outcomes of filovirus infections between bats and humans. In this study, we outline multiple interconnected pathways that respond to infection by MARV and EBOV, providing insights into the complexity of the mechanisms that enable bats to resist the disease caused by filoviral infections. The results have the potential to aid in the development of new strategies to effectively mitigate and treat the disease caused by these viruses in humans. Improved AlphaFold modeling with implicit experimental information Machine learning prediction algorithms such as AlphaFold1 and RoseTTAFold2 can create remarkably accurate protein models, but these models usually have some regions that are predicted with low confidence or poor accuracy3-6. We hypothesized that by implicitly including experimental information, a greater portion of a model could be predicted accurately, and that this might synergistically improve parts of the model that were not fully addressed by either machine learning or experiment alone. An iterative procedure was developed in which AlphaFold models are automatically rebuilt based on experimental density maps and the rebuilt models are used as templates in new AlphaFold predictions. We find that including experimental information improves prediction beyond the improvement obtained with simple rebuilding guided by the experimental data. This procedure for AlphaFold modeling with density has been incorporated into an automated procedure for crystallographic and electron cryo-microscopy map interpretation. Lysophosphatidyl-choline 16:0 mediates persistent joint pain through Acid-Sensing Ion Channel 3: preclinical and clinical evidences Rheumatic diseases are often associated to debilitating chronic joint pain, which remains difficult to treat and requires new therapeutic strategies. Here, we describe increased content of lysophosphatidyl-choline (LPC) 16:0 in the knee synovial fluids of two independent cohorts of patients with painful joint diseases. If LPC16:0 levels correlated with pain in patients with osteoarthritis (OA), they do not appear to be the hallmark of a particular joint disease. We found that intra-articular injections of LPC16:0 in mouse produce chronic pain and anxiety-like behaviors in both males and females with no apparent inflammation, peripheral nerve sprouting and damage, nor bone alterations. LPC16:0-induced persistent pain state is dependent on peripheral Acid-Sensing Ion Channel 3 (ASIC3), ultimately leading to central sensitization. LPC16:0 and ASIC3 thus appear as key players of chronic joint pain with potential implications in OA and possibly across others rheumatic diseases. Engineered bacteria detect tumor DNA Advances in bacterial engineering have catalysed the development of living cell diagnostics and therapeutics1-3, including microbes that respond to diseases such as gut inflammation4, intestinal bleeding5, pathogens6 and hypoxic tumors7. Bacteria can easily access the entire gastrointestinal tract via oral administration8, and they can produce outputs that can be noninvasively measured in stool4 or urine7. Cellular memory, such as bistable switches4,9,10 or genomic rearrangement11, has been used to allow bacteria to store information over time. However, living biosensors have not yet been engineered to detect specific DNA sequences or mutations from outside the cell. Here, we engineer naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells and organoids. We characterize the functionality of the biosensors in vitro with co-culture assays and then validate in vivo with sensor bacteria delivered orally or rectally into mice injected with orthotopic donor CRC organoids. We observe horizontal gene transfer from the tumor to the sensor bacteria in vivo, allowing their detection in stool. The sensor bacteria achieved 100% discrimination between mice with and without CRC using both delivery methods. Our findings establish a framework for biosensing applications that require the detection of mutations or organisms within environments that are difficult to sample. In addition, the platform can be readily expanded to include in situ production and delivery of therapeutic payloads at the detection site. Age of the bone marrow dictates clonality of smooth muscle-derived cells in the atherosclerotic plaque Aging is the predominant risk factor for atherosclerosis, the leading cause of death. Rare smooth muscle cell (SMC) progenitors clonally expand giving rise to up to [~]70% of atherosclerotic plaque cells; however, the effect of age on SMC clonality is not known. Our results indicate that aged bone marrow (BM)-derived cells non-cell autonomously induce SMC polyclonality and worsen atherosclerosis. Indeed, in myeloid cells from aged mice and humans, TET2 levels are reduced which epigenetically silences integrin {beta}3 resulting in increased tumor necrosis factor [TNF]- signaling. In turn, TNF signals through TNF receptor 1 on SMCs to promote proliferation and induces recruitment and expansion of multiple SMC progenitors into the atherosclerotic plaque. Notably, integrin {beta}3 overexpression in aged BM preserves dominance of the lineage of a single SMC progenitor and attenuates plaque burden. Our results demonstrate a molecular mechanism of aged macrophage-induced SMC polyclonality and atherogenesis and suggest novel therapeutic strategies.

Graphical abstractAge of BM-derived monocytes/macrophages determines clonality of SMC lineage in the atherosclerotic plaque. Atherogenesis is depicted in a young (a) or aged (b) host. Aged monocytes/macrophages have decreased levels of the epigenetic regulator TET2, leading to reduction of the 5-hydroxymethylcytosine (5hmC) mark on the Itgb3 promoter. The resulting low integrin {beta}3 levels in aged monocytes/macrophages induces high TNF- levels, facilitating recruitment and expansion of multiple SMC progenitors (polyclonality) in the atherosclerotic plaque and worse disease burden. In contrast, the young control is characterized by mono/oligoclonal SMC expansion in a smaller plaque.



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org.highwire.dtl.DTLVardef@12f7090org.highwire.dtl.DTLVardef@186ff92org.highwire.dtl.DTLVardef@1f4da69org.highwire.dtl.DTLVardef@2f509c_HPS_FORMAT_FIGEXP  M_FIG C_FIG An epigenetic memory of inflammation controls context-dependent lineage plasticity in the pancreas Inflammation is essential to the disruption of tissue homeostasis, and, in the pancreas, can destabilize the identity of terminally differentiated acinar cells. Herein we employ lineage-traced mouse models to delineate the chromatin dynamics that accompany the cycle of metaplasia and regeneration following pancreatitis, and unveil the presence of an epigenetic memory of inflammation in the pancreatic acinar cell compartment. We observe that despite histologic resolution of pancreatitis, acinar cells fail to return to their molecular baseline after several months, representing an incomplete cell fate decision. In vivo, this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second inflammatory insult but increased tumorigenesis with an oncogenic Kras mutation. We demonstrate that both persistent chromatin and transcriptional changes constituting memory are recalled with oncogenic stress. Together, our findings define the dynamics and recall of an epigenetic memory of inflammation that impacts cell fate decisions in a context-dependent manner. Chromosome counting in the mouse zygote using low-invasive super-resolution live-cell imaging In preimplantation embryos, an abnormal chromosome number causes developmental failure and a reduction in the pregnancy rate. Conventional chromosome testing methods requiring biopsy reduce the risk of associated genetic diseases; nevertheless, the reduction in cell number also reduces the pregnancy rate. Therefore, we attempted to count the chromosomes in mouse (Slc:ICR) embryos using super-resolution live-cell imaging as a new method of chromosome counting that does not reduce the cell number or viability. We counted the forty chromosomes at the first mitosis by injecting embryos with histone H2B-mCherry mRNA under conditions by which pups could be obtained; however, the results were often an underestimation of chromosome number and varied by embryo and time point. Therefore, we developed a method to count the chromosomes via CRISPR/dCas-mediated live-cell fluorescence in situ hybridization targeting the sequence of the centromere region, enabling us to count the chromosomes more accurately in mouse embryos. The methodology presented here may provide useful information for assisted reproductive technologies, such as those used in livestock animals/humans, as a technique for assessing the chromosomal integrity of embryos prior to transfer.

Impact StatementLow-toxic super-resolution observation enables chromosome counting in preimplantation embryos without cell collection. Allopatric divergence of cooperators confers cheating resistance and limits the effects of a defector mutation Social defectors may meet diverse cooperators. Genotype-by-genotype interactions may constrain the ranges of cooperators upon which particular defectors can cheat, limiting cheater spread. The bacterium Myxococcus xanthus undergoes cooperative multicellular development, but some developmental defectors cheat on cooperators during this process. In this study, interactions between a cheater disrupted at the signaling gene csgA and allopatrically diversified cooperators reveal a very small cheating range. Expectedly, the cheater failed to cheat on all natural-isolate cooperators owing to non-cheater-specific antagonisms. Surprisingly, lab-evolved cooperators that diverged from their cheating-susceptible ancestor by fewer than 20 mutations and without experiencing cheating had already exited the csgA mutants cheating range. Cooperators might also diversify in the potential for a mutation to reduce expression of cooperative trait or generate a cheating phenotype. A new csgA mutation constructed in several highly diverged cooperators generated diverse sporulation phenotypes, ranging from a complete defect to no defect, indicating that genetic backgrounds can limit the set of genomes in which a mutation creates a defector. Our results suggest that natural populations feature geographic mosaics of cooperators that have diversified in their susceptibility to particular cheaters and in the phenotypes generated by any given cooperation-gene mutation.

Significance statementSelection on cooperators exploited by obligate cheaters can induce evolution of resistance to cheating. Here we show that cooperators can also rapidly evolve immunity to cheating simply as a byproduct of evolutionary divergence in environments in which cooperation and cheating at the focal trait do not occur because the trait is not expressed. We also find that differences in the genomic context in which a cooperation-gene mutation arises can profoundly alter its phenotypic effect and determine whether the mutation generates a social defect at all - a pre-requisite for obligate cheating. These findings suggest that general divergence of social populations under a broad range of environmental conditions can restrict both the set of mutations that might generate social defectors in the first place and the host range of such defectors once they arise. Discoidin Domain Receptor 2 regulates AT1R expression in Angiotensin II-stimulated cardiac fibroblasts via fibronectin-dependent Integrin-β1 signaling Recent reports on the cardioprotective effects of fibronectin inhibition following myocardial injury suggest a largely unexplored role for the extracellular matrix (ECM) glycoprotein in cardiac fibroblast function. We probed the molecular regulation and functional implications of fibronectin gene expression in cardiac fibroblasts exposed to Angiotensin II, a major pro-fibrotic factor in the myocardium. Using gene knockdown and over-expression approaches, western blotting and promoter pull-down assay, we show that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Angiotensin II-stimulated transcriptional up-regulation of fibronectin by Yes-activated Protein in cardiac fibroblasts. Further, siRNA-mediated fibronectin knockdown attenuated Angiotensin II-stimulated expression of collagen type I and anti-apoptotic cIAP2, and enhanced susceptibility to apoptosis. Importantly, an obligate role for fibronectin was observed in Angiotensin II-stimulated expression of AT1R, the Angiotensin II receptor, which would link ECM signaling and Angiotensin II signaling in cardiac fibroblasts. Moreover, conditioned medium from DDR2- or fibronectin-silenced cardiac fibroblasts reduced AT1R expression in H9c2 cardiomyoblasts. The regulatory role of fibronectin in Angiotensin II-stimulated cIAP2, collagen type I and AT1R expression was mediated by Integrin-{beta}1-integrin-linked kinase signaling. In vivo, we observed modestly reduced basal levels of AT1R in DDR2-null mouse myocardium, associated with the previously reported reduction in myocardial Integrin-{beta}1 levels. The role of fibronectin, downstream of DDR2, could be a critical determinant of cardiac fibroblast-mediated wound healing following myocardial injury. In summary, our findings suggest a complex mechanism of regulation of cardiac fibroblast function involving two major extracellular matrix proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-{beta}1. 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. Design of Specific Primer Sets for the Detection of B.1.1.7, B.1.351, P.1, B.1.617.2 and B.1.1.519 Variants of SARS-CoV-2 using Artificial Intelligence As the COVID-19 pandemic continues, new SARS-CoV-2 variants with potentially dangerous features have been identified by the scientific community. Variant B.1.1.7 lineage clade GR from Global Initiative on Sharing All Influenza Data (GISAID) was first detected in the UK, and it appears to possess an increased transmissibility. At the same time, South African authorities reported variant B.1.351, that shares several mutations with B.1.1.7, and might also present high transmissibility. Earlier this year, a variant labelled P.1 with 17 non-synonymous mutations was detected in Brazil. Recently the World Health Organization has raised concern for the variants B.1.617.2 mainly detected in India but now exported worldwide. It is paramount to rapidly develop specific molecular tests to uniquely identify new variants. Using a completely automated pipeline built around deep learning and evolutionary algorithms techniques, we designed primer sets specific to variants B.1.1.7, B.1.351, P.1 and respectively. Starting from sequences openly available in the GISAID repository, our pipeline was able to deliver the primer sets for each variant. In-silico tests show that the sequences in the primer sets present high accuracy and are based on 2 mutations or more. In addition, we present an analysis of key mutations for SARS-CoV-2 variants. Finally, we tested the designed primers for B.1.1.7 using RT-PCR. The presented methodology can be exploited to swiftly obtain primer sets for each new variant, that can later be a part of a multiplexed approach for the initial diagnosis of COVID-19 patients. Collective polymerase dynamics emerge from DNA supercoiling during transcription All biological processes ultimately come from physical interactions. The mechanical properties of DNA play a critical role in transcription. RNA polymerase can over or under twist DNA (referred to as DNA supercoiling) when it moves along a gene resulting in mechanical stresses in DNA that impact its own motion and that of other polymerases. For example, when enough supercoiling accumulates, an isolated polymerase halts and transcription stops. DNA supercoiling can also mediate non-local interactions between polymerases that shape gene expression fluctuations. Here, we construct a comprehensive model of transcription that captures how RNA polymerase motion changes the degree of DNA supercoiling which in turn feeds back into the rate at which polymerases are recruited and move along the DNA. Surprisingly, our model predicts that a group of three or more polymerases move together at a constant velocity and sustain their motion (forming what we call a polymeton) whereas one or two polymerases would have halted. We further show that accounting for the impact of DNA supercoiling on both RNA polymerase recruitment and velocity recapitulates empirical observations of gene expression fluctuations. Finally, we propose a mechanical toggle switch whereby interactions between genes are mediated by DNA twisting as opposed to proteins. Understanding the mechanical regulation of gene expression provides new insights into how endogenous genes can interact and informs the design of new forms of engineered interactions.

PACS numbers: Long-read and chromosome-scale assembly of the hexaploid wheat genome achieves high resolution for research and breeding The sequencing of the wheat (Triticum aestivum) genome has been a methodological challenge for many years due to its large size (15.5 Gb), repeat content, and hexaploidy. Many initiatives aiming at obtaining a reference genome of cultivar Chinese Spring have been launched in the past years and it was achieved in 2018 as the result of a huge effort to combine short-read sequencing with many other resources. Reference-quality genome assemblies were then produced for other accessions but the rapid evolution of sequencing technologies offers opportunities to reach high-quality standards at lower cost. Here, we report on an optimized procedure based on long-reads produced on the ONT (Oxford Nanopore Technology) PromethION device to assemble the genome of the French bread wheat cultivar Renan. We provide the most contiguous and complete chromosome-scale assembly of a bread wheat genome to date. Coupled with an annotation based on RNA-Seq data, this resource will be valuable for the crop community and will facilitate the rapid selection of agronomically important traits. We also provide a framework to generate high-quality assemblies of complex genomes using ONT. Distributed brain co-processor for tracking electrophysiology and behavior during electrical brain stimulation Early implantable epilepsy therapy devices provided open-loop electrical stimulation without brain sensing, computing, or an interface for synchronized behavioral inputs from patients. Recent epilepsy stimulation devices provide brain sensing but have not yet developed analytics for accurately tracking and quantifying behavior and seizures. Here we describe a distributed brain co-processor providing an intuitive bi-directional interface between patient, implanted neural stimulation and sensing device, and local and distributed computing resources. Automated analysis of continuous streaming electrophysiology is synchronized with patient reports using a hand-held device and integrated with distributed cloud computing resources for quantifying seizures, interictal epileptiform spikes, and patient symptoms during therapeutic electrical brain stimulation. The classification algorithms for interictal epileptiform spikes and seizures were developed and parameterized using long-term ambulatory data from 9 humans and 8 canines with epilepsy, and then implemented prospectively in out-of-sample testing in 2 pet canines and 4 humans with drug resistant epilepsy living in their natural environments. Social integration predicts survival in female white-faced capuchin monkeys Across multiple species of social mammals, a growing number of studies have found that that individual sociality is associated with survival. In long-lived species, like primates, lifespan is one of the main components of fitness. We used 18 years of data from the Lomas Barbudal Monkey project to quantify social integration in 11 capuchin (Cebus capucinus) groups and tested whether female survivorship was associated with females tendencies to interact with three types of partners: (1) all group members, (2) adult females, (3) adult males. We found strong evidence that females who engaged more with other females in affiliative interactions and foraged in close proximity experienced increased survivorship. We found some weak evidence that females might also benefit from engaging in more support in agonistic contexts with other females. These benefits were evident in models that account for the females rank and group size. Female interactions with all group members also increased survival, but the estimates of the effects were more uncertain. In interactions with adult males, only females who provided more grooming to males survived longer. The results presented here suggest that social integration may result in survival-related benefits. Females might enjoy these benefits through exchanging grooming for other currencies, such as coalitionary support or tolerance. Trehalose increases tomato drought tolerance, induces defenses, and increases resistance to bacterial wilt disease Ralstonia solanacearum causes bacterial wilt disease, leading to severe crop losses. Xylem sap from R. solanacearum-infected tomato is enriched in the disaccharide trehalose. Water-stressed plants also accumulate trehalose, which increases drought tolerance via abscisic acid (ABA) signaling. Because R. solanacearum-infected plants suffer reduced water flow, we hypothesized that bacterial wilt physiologically mimics drought stress, which trehalose could mitigate. We found that R. solanacearum-infected plants differentially expressed drought-associated genes, including those involved in ABA and trehalose metabolism, and had more ABA in xylem sap. Consistent with this, treating tomato roots with ABA reduced both stomatal conductance and stem colonization by R. solanacearum. Treating roots with trehalose increased xylem sap ABA and reduced plant water use by lowering stomatal conductance and temporarily improving water use efficiency. Trehalose treatment also upregulated expression of salicylic acid (SA)-dependent tomato defense genes; increased xylem sap levels of SA and other antimicrobial compounds; and increased bacterial wilt resistance of SA-insensitive NahG tomato plants. Additionally, trehalose treatment increased xylem concentrations of jasmonic acid and related oxylipins. Finally, trehalose-treated plants were substantially more resistant to bacterial wilt disease. Together, these data show that exogenous trehalose reduced both water stress and bacterial wilt disease and triggered systemic disease resistance, possibly through a Damage Associated Molecular Pattern (DAMP) response pathway. This suite of responses revealed unexpected linkages between plant responses to biotic and abiotic stress and suggested that R. solanacearum-infected plants increase trehalose to improve water use efficiency and increase wilt disease resistance. The pathogen may degrade trehalose to counter these efforts. Together, these results suggest that treating tomatoes with exogenous trehalose could be a practical strategy for bacterial wilt management. Comparison of hybrid learning and remote education in the implementation of the 'Adopt a Microorganism' methodology The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. 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. Crowdsourced identification of multi-target kinase inhibitors for RET- and TAU- based disease: the Multi-Targeting Drug DREAM Challenge A continuing challenge in modern medicine is the identification of safer and more efficacious drugs. Precision therapeutics, which have one molecular target, have been long promised to be safer and more effective than traditional therapies. This approach has proven to be challenging for multiple reasons including lack of efficacy, rapidly acquired drug resistance, and narrow patient eligibility criteria. An alternative approach is the development of drugs that address the overall disease network by targeting multiple biological targets ( polypharmacology). Rational development of these molecules will require improved methods for predicting single chemical structures that target multiple drug targets. To address this need, we developed the Multi-Targeting Drug DREAM Challenge, in which we challenged participants to predict single chemical entities that target pro-targets but avoid anti-targets for two unrelated diseases: RET-based tumors and a common form of inherited Tauopathy. Here, we report the results of this DREAM Challenge and the development of two neural network-based machine learning approaches that were applied to the challenge of rational polypharmacology. Together, these platforms provide a potentially useful first step towards developing lead therapeutic compounds that address disease complexity through rational polypharmacology.

Author SummaryMany modern drugs are developed with the goal of modulating a single cellular pathway or target. However, many drugs are, in fact,  dirty; they target multiple cellular pathways or targets. This phenomenon is known as multi-targeting or polypharmacology. While some strive to develop  cleaner therapeutics that eliminate secondary targets, recent work has shown that multi-targeting therapeutics have key advantages for a variety of diseases. However, while multi-targeting drugs that affect a precisely-defined profile of targets may be more effective, it is difficult to computationally predict which molecules have desirable target profiles. Here, we report the results of a competitive crowdsourcing project (the Multi-Targeting Drug DREAM Challenge), where we challenged participants to predict chemicals that have desired target profiles for cancer and neurodegenerative disease. 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. A natural mutator allele shapes mutation spectrum variation in mice Summary ParagraphAlthough germline mutation rates and spectra can vary within and between species, genetic modifiers of these traits have long eluded detection. In this study, we searched for loci that influence germline mutagenesis using a uniquely powerful resource: a panel of recombinant inbred mouse lines known as the BXD, descended from the laboratory mouse strains C57BL/6J (B) and DBA/2J (D). Each BXD lineage has been maintained by brother-sister mating in the near absence of natural selection, accumulating de novo mutations for up to 50 years on a known genetic background that is a unique linear mosaic of B and D haplotypes. We show that mice inheriting D haplotypes at a quantitative trait locus (QTL) on chromosome 4 accumulate C>A germline mutations at a 50% higher rate than those inheriting B haplotypes, primarily due to the activity of a C>A-dominated mutational signature known as SBS18. The B and D QTL haplotypes encode different alleles of the DNA repair gene Mutyh, which underlies the heritable colorectal cancer syndrome in which SBS18 was first identified. The B and D Mutyh alleles are present in wild populations of Mus musculus domesticus, providing evidence that common genetic variation modulates germline mutagenesis in a model mammalian species. HSP90 modulates T2R bitter taste receptor nitric oxide production and innate immune responses in human airway epithelial cells and macrophages Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two airway innate immune cell types are activated by bitter products, including some secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells and increases phagocytosis by macrophages. Using biochemistry and live cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). We used immunofluorescence to show that H441 cells express eNOS and certain T2Rs and that the bitterant denatonium benzoate activates NO production in an HSP90-dependent manner in cells grown either as submerged cultures and at air liquid interface. In primary sinonasal epithelial cells, we determined that HSP-90 inhibition reduces T2R-stimulated NO production and ciliary beating which are crucial for pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves an innate immune role by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream calcium signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases like chronic rhinosinusitis, asthma, or cystic fibrosis. The ChAdOx1 vectored vaccine, AZD2816, induces strong immunogenicity against SARS-CoV-2 Beta (B.1.351) and other variants of concern in preclinical studies. There is an ongoing global effort to design, manufacture, and clinically assess vaccines against SARS-CoV-2. Over the course of the ongoing pandemic a number of new SARS-CoV-2 virus isolates or variants of concern (VoC) have been identified containing mutations in key proteins. In this study we describe the generation and preclinical assessment of a ChAdOx1-vectored vaccine (AZD2816) which expresses the spike protein of the Beta VoC (B.1.351). We demonstrate that AZD2816 is immunogenic after a single dose. When AZD2816 is used as a booster dose in animals primed with a vaccine encoding the original spike protein (ChAdOx1 nCoV-19/ [AZD1222]), high titre binding and neutralising antibodies against Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) are induced. In addition, a strong and polyfunctional T cell response was measured in these booster regimens. These data support the ongoing clinical development and testing of this new variant vaccine.